JP2009523175A - Pharmaceutical compositions and methods for preventing and treating copper-related central nervous system diseases by achieving and maintaining a targeted stable copper state - Google Patents

Abstract

Compositions and methods comprising improved means for achieving and maintaining a targeted level of copper status for treating and preventing copper-related diseases in humans.
[Selection figure] None

Description

  The present invention relates to pharmaceutical products and methods for treating excessive metal accumulation or metal malabsorption in animals and humans. The present invention is particularly suitable for the treatment of human Wilson's disease and describes its use, but excessive accumulation of copper in the brain and other organs and / or free copper in the serum and central nervous system (CNS). It can also be applied to the treatment of neurological diseases caused by changes in the daily value of copper). In addition to, but not limited to, central nervous system diseases, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Lou Gehrig's disease, dementia, Huntington's disease, schizophrenia, Examples include neuromuscular diseases associated with abnormal accumulation of copper-related proteins (e.g. juvenile and sporadic inclusion body myositis, elderly myositis, and heart diseases such as atherosclerosis, cerebral infarction and peripheral blood vessels). it can. The present invention relates to neurological and psychiatric symptoms of liver diseases associated with reduced liver copper excretion (e.g. cholangitis, hepatitis and cirrhosis in which free or weakly bound serum copper or CSF copper increases). It can also be used to treat.

  Copper is an essential trace element for life. However, despite its importance, copper is also a highly reactive oxidant and is highly toxic to cells, proteins and organ systems (eg, liver, brain and vasculature). In order to deliver and utilize copper in the body, where needed, in mammalian systems, as required, with the ability of copper to inadvertently oxidize or reduce proteins and lipids, A close control network has been developed for a highly specific, homeostatic copper-binding copper protein that has the effect of properly removing, storing, transporting, chaperoning and excreting copper. Many different copper proteins have been identified and their functions have been elucidated over the years. Examples of such copper proteins include, but are not limited to, matrix metalloprotein, ceruloplasmin, copper / zinc superoxide dismutase, amyloid precursor protein, apolipoprotein E, tau (τ), homo There are chaperones for cysteine, albumin and copper zinc superoxide dismutase.

One of the potential toxic sources where copper for humans is most problematic is the presence of large amounts of harmful copper in the drinking water system. This is because copper present in food is bound to protein and its absorption can be regulated relatively easily in the intestine, and it slowly degrades from food during digestion, whereas copper in drinking water It is in the form of copper ions (Cu ⁺² ), in a non-bonded form or only weakly complexed with organic ligands. Copper ions are generally more bioavailable in water than in food. In the human diet, there are components in the food that affect copper metabolism, absorption and fluidization.

<Copper absorption by human body>
In humans, dietary copper is absorbed from the stomach and small intestine. Approximately 65% of the oral dose of Cu ⁶⁴ as copper sulfate was absorbed from the human gastrointestinal tract (15-97%) [Weber et al., 1969; Strickland et al., 1972]. It is inversely related to copper content in food [Turnland et al., 1989, 1998]. Orally administered Cu ⁺⁶⁴ appears rapidly in plasma [Beam and Kunkel, 1955].

When human full-term infants consumed 114 μg / kg-day of copper daily with breast milk, 88 μg / kg-day of copper remained. This represents an absorption value of approximately 77% [Dorner et al. (1989)]. Copper residue decreased with age. At 2 weeks after birth, 130 μg / kg-day remained, and at 16 weeks after birth, 64 μg / kg-day remained. In contrast, the average relative retention of infants given the copper reinforcement formulation was 52 percent. Copper absorption in red-haired monkey infants using Cu ⁶⁷ was 50-70 percent, similar to that of human full-term infants [Lonnerdal et al., 1996]. According to a study in rats, copper absorption was very high during neonates but decreased at weaning [Lonnerdal et al., 1985]. Perfusion of the rat intestine [Varada et al. (1993), copper absorption was linear and non-saturated in infants and weaners, but saturated in adolescent rats. In infant rats, it was significantly higher than weanling or adolescent rats, and citrate present in human and cow milk has been shown to have an improving effect on copper absorption in animal models [Shah, 1981] .

An oral supplement 80 μg Cu (as a copper sulfate solution) / kg was administered daily to a Chilean 1-3 month (n = 20) infant group [Olivares et al. (2002)]. Half of the infant group (n = 19) did not receive supplements. At the end of the study, Cu ⁶⁵ was orally administered as a tracer, stool was monitored for the recovered Cu ⁶³ , copper absorption was measured, and no significant difference was observed in the absorbed copper between the two infant groups It was. The average copper absorption (+ SD) at 1 month of age was 83.6 + 5.8 percent with supplements and 74.8 + 15.2 percent with no supplements. The report concludes that the experimental design was inadequate because copper intake was too low to “trigger the adaptation due to homeostasis of intestinal absorption”.

  The absorption of copper in the digestive tract of rats and hamsters was studied. Absorption occurred from the stomach and duodenum in rats [Van Campen and Mitchell, 1965], and in hamsters from the lower small intestine [Crampton et al., 1965]. Copper absorbed from the gastrointestinal tract can be bound to amino acids or can exist in the form of ionic copper. Copper is bound to the metallothionein in the intestine and either absorbed in the intestine or returned and discarded.

  The presence of a protein source (plant protein or animal protein), amino acids, carbohydrates and / or ascorbic acid affects the availability of copper [Gibson, 1994; Lonnerdal, 1996]. Antagonism of zinc and cadmium affects copper absorption from both diet and drinking water [Davies and Campbell, 1977; Hall et al., 1979]. Ascorbic acid can change the binding site of metallothionein. In guinea pigs, dietary ascorbic acid has been shown to interfere with copper absorption [Smith and Bidlack, 1980]. However, in humans, it has been shown that normal doses of ascorbic acid are not a factor [Jacob et al., 1987]. The phytate and fiber form a complex with copper and prevent copper absorption [Gibson, 1994]. In humans, copper accumulates primarily in the liver but does not affect copper absorption [Strickland et al., 1972]. There are no studies available on the absorption of copper by inhalation in humans.

  After rats were exposed to pure copper wire welding dust, copper oxide in alveolar capillaries was observed [Batsura, 1969]. Although no study has been conducted on the rate or extent of copper absorption through human original skin, some absorption occurs because copper causes contact dermatitis [ATSDR, 1990]. There is a study on in vitro absorption of copper and zinc through human skin [Pirot et al. (1996). Skin absorption does not contribute significantly to total copper absorption.

<Distribution of copper in the human body>
Copper is transported through the plasma and bound to ceruloplasmin, albumin or amino acids [Cousins, 1985]. Ceruloplasmin is a glycoprotein rich in cysteine, and many free sulfhydryl groups function as binding sites for metals. Ceruloplasmin can bind copper or zinc, but has a stronger binding to copper [Cousins, 1985]. Ceruloplasmin is synthesized in the membrane-bound polyribosomes of liver parenchyma cells and secreted into the plasma. Copper entering the portal circulation from the intestine is transported directly to the liver. Copper released from the liver is transported through the bloodstream to other organs (including the kidneys and brain). Ceruloplasmin synthesis is regulated by interleukin-I via glucagon or glucocorticoid [Cousins, 1985]. The amount of circulating copper is increased in pregnant women because hormonal changes associated with pregnancy stimulate the synthesis of ceruloplasmin [Solomons, 1985]. Ceruloplasmin value is useful as an indicator of copper status [Mendez et al., 2004].

  Recently, several copper transporters have been identified that are involved in the uptake and transport of copper by cells [Bauerly et al., 2005]. Copper transporter-1 (Crt1) is a copper-specific copper uptake protein and is thought to regulate copper uptake into the small intestine [Lee et al., 2002]. Crt1 is expressed in the enterocytes of the small intestine and in the enterocyte-like Caco-2 of the culture [Klomp et al., 2002; Kuo et al., 2001]. The copper efflux protein ATP7A is thought to regulate the efflux of copper through the plasma membrane during copper expression in transfected cells [Petris et al., 1996]. Menkes disease is characterized by excessive copper accumulation in the small intestine and systemic copper deficiency and is the result of ATP7A deficiency [Schaefer and Gitlin, 1999]. ATP7B is functionally similar to ATP7A and exports copper into the bile for excretion [Roelofsen et al., 2000]. ATP7B is mainly present in the liver and is less expressed in the small intestine, kidney and placenta [Lockhart et al., 2000]. Abnormalities in ATP7B result in Wilson disease, which is characterized by copper toxicity (due to hepatic copper accumulation as a result of reduced biliary copper excretion) and liver damage.

<Metabolism and discharge of copper>
The liver and intestine play an important role in copper metabolism. Copper is taken up by hepatocytes from the portal circulation. In hepatocytes, copper is bound to metallothionein. Metallothionein is a protein that also binds copper, iron and mercury. Copper is released from the hepatocytes into the systemic circulation and transported to other tissues or excreted from the liver into bile [Cousins, 1985]. The main destination is in bile. Very small amounts are excreted in the urine [Cousins, 1985]. Human infants are underdeveloped at the time of birth, and lack of effective drainage functions increases the risk of infants to copper toxicity.

<Physiological / nutritional role of copper>
Because copper is an essential nutrient, understanding the many physiological roles in the body is important in understanding the deleterious effects of copper overs and shorts. Copper is important for hemoglobin synthesis and red blood cell production [Solomons, 1985; Harris, 1997]. Copper shortage leads to anemia. Copper deficiency also results in myelin production abnormalities and incidental effects on the nervous system [Solomons, 1985; Harris, 1997]. As an effect on the nervous system, for example, dementia was observed in individuals with copper deficiency [Solomons, 1985; Harris, 1997]. The effect on catecholamine metabolism is similarly related to nervous system abnormalities. Other physiological functions associated with copper include leukogenesis, skeletal calcification, connective tissue synthesis, melanin synthesis, oxidative phosphorylation, thermoregulation, antioxidant protection, cholesterol metabolism, immune and cardiac function and regulation of glucose metabolism Can be mentioned. Since all these physiological processes are related to copper, all are affected by the availability of copper in the human body or specific tissues. In general, any of these physiological processes can be adversely affected in systems affected by copper deficiency [Solomons, 1985; Harris, 1997].

  The specific requirements for copper for infants are not well documented. In infants, copper is an essential mineral for normal growth of bone, brain, immune system, and red blood cells [Hurley et al., 1980]. Full-term infants have the right amount of copper at birth and are thought to continue until weaning, but premature infants are prone to copper shortages and supply more copper to compensate for the shortage Need to do [Lonnerdal, 1998].

  For copper, the recommended daily intake (RDAs) was not specified in the initial version of RDA because it was difficult to determine the value [NAS, 1989]. When eating food, the homeostasis mechanism changes the absorption and excretion of copper, which complicates the calculation of mass balance when considering nutrition. However, many recent publications have recommended amounts [FNB, 2000] and copper nutritional conditions. Table 1 below shows the estimated average requirement (EAR), nutritional requirement (RDA), and maximum allowable intake (UL) for dietary intake standards (DRI) for copper in various age groups [FNB, From 2000]. Infants are listed as appropriate intake (AI) values based mainly on the amount of copper contained in human milk. AI values were 200 μg / day for infants 0-6 months and 220 μg / day for infants 7-12 months, and the UL estimate for infants could not be specified [FNB, 2000].

  Copper intake from food and supplements was obtained from the NHANES III national survey (1988-1994) and is shown in Table 2 below. The NHANES III table and the continuous survey of food intake (CSFII) show adequate copper intake for most of all ages and gender groups. However, results for some younger groups show that as much as 10% of the population consumes less than copper RDA. On the other hand, the lower end of the distribution curve seems inaccurate, especially considering the tendency of teenagers to underreport food intake [Champagne et al., 1998].

In Table 2, for RDA, breastfeeding infants and children and 8-year-olds who reported more than 150 mg / day of copper from supplements were excluded from the analysis. The value of RDA is the value of FNB, 2000.
Although most people can cope with chronic exposure to toxic copper ions in drinking water without showing signs of illness, the effects of genetic variants on human copper transport and metabolic pathways Infrequent illnesses (eg Wilson disease or Menkes disease) occur rarely. Several groups have confirmed that genetic variants cause Wilson and Menkes disease in the early 1990s. In addition to rare Wilson disease patients, serum copper levels have been reported in older adults [Madaric et. Al., Physiol Res, 1994; 43 (2): 107-11 and Ghayour-Mobarhan et. Al. , Ann Clin Biochem, 2005 Sep; 42 (Pt 5): 364-75]. This may be due to an impairment in the ability of copper to be properly processed for delivery to the bile through the liver and excreted as feces. Therefore, this patient population may have copper sensitivity similar to Wilson's disease patients. Older Alzheimer's disease patients have been reported to have increased levels of non-ceruloplasmin-bound copper [Squitti, et. Al.].

  Wilson's disease is characterized by a mutation in a gene encoding a P-type ATPase called ATP7B. Due to ATP7B deficiency, patients with Wilson disease are unable to properly handle copper and transport and excrete copper through the normal bile ducts of the liver. In normal cases, newly introduced copper appears to bind first to available endogenous copper proteins with the highest affinity for copper (e.g., metallothionein, superoxide dismutase and albumin). It is. In the case of copper ions, there are many coppers in Wilson's disease patients that are fairly rare in serum but are “weakly bound” to various proteins and peptides. This is also potentially applicable to Alzheimer's patients with other metabolic disorders, mild cognitive impairment (MCI) patients, schizophrenia patients, dementia patients and the elderly.

  In the 1990s, the genetic defect responsible for Menkes disease was identified as a mutation to another P-type ATPase, ATP7A. Menkes disease is characterized by the inability of intestinal cells to release copper, resulting in abnormally low levels of available copper and various developmental abnormalities.

  Estimates of the contribution to total serum copper content in various serum proteins in normal patients are as follows. Ceruloplasmin (650-750μg / L, 65-70%), albumin (120-180μg / L, 12-18%), transcuplein (macroglobulin) (90μg / L, 9%), ferroxidase II (10μg / L, 1%), extracellular SOD and histidine-enriched glycoprotein (<10 μg / L, <1%), blood coagulation factors V and VIII (<5 μg / L, <0.5%), extracellular metallothionein and anime oxidase (<1 μg / L, <0.1%), 15-6 OkDa component (40 μg / L, 4%), small peptides and amino acids (35 μg / L, 4%), unbound or free copper ions (0.0001 μg / L, About 0%) [Linder, MC Biochemistry of Copper (ed.) 1991; Linder, MC (2001), Copper and Genomic Stability in Mammals., Mutat. Res. 475, 151-52].

  As mentioned above, the majority of circulating serum copper is bound to 15-60 kDa protein (about 4%) and small peptides and amino acids (further about 4%). Such small proteins and peptides can pass weakly bound copper through the blood brain barrier, creating an environment where copper can be present in excess and detrimental to neuronal health. In order to maintain healthy copper homeostasis and protect against extracellular lipid superoxidation and intracellular oxidation, neurons can up-regulate various copper binding proteins. These copper binding proteins include, for example, APP, amyloid beta, tau, BACEl, and apoE, all of which are up-regulated in Alzheimer's disease, and also in cells and in inclusion body myositis of neuromuscular diseases.

  Dissolved copper or copper that is weakly bound to small ligands is generally present in tap water, but these copper are highly bioavailable (up to 65%) and are digested by water fluxes in the intestines. Has ability to overwhelm copper intestinal cells and hepatic copper homeostasis and enters portal vein and systemic circulation in a weakly binding form potentially detrimental to albumin and other low kinetic copper binding proteins . The object of the present invention is to provide compositions, formulations, agents and methods for protecting against such harmful flux, as described below.

<Wilson's disease>
In untreated patients with Wilson disease, copper in the body continues to accumulate, eventually overwhelming the high-binding copper protein. Residual copper is weakly bound to copper proteins that are free or unbound or have low binding to copper. This pool of free, unbound or low binding copper is free to circulate and can cross the blood brain barrier, thus impairing neurons due to its reactivity and pro-oxidative capacity. Clinically, various copper proteins act as copper reservoirs and are generally released based on an inverse correlation with the binding properties of each copper protein to copper. Examples of so-called “loosely bound” copper proteins include albumin and homocysteine. Such copper proteins act as a potentially toxic pool of available copper compared to proteins with strong copper binding (eg, ceruloplasmin and Cu / Zn superoxide dismutase).

  When copper excretion is impaired due to genetic disease or liver damage due to fibrosis or bile flow, highly binding copper proteins such as metallothionein eventually become fully saturated, The copper binding process continues with proteins with low copper binding properties such as homocysteine and albumin, and each of these protein pools becomes saturated. In patients with Wilson's disease, total copper overwhelms the ability of high binding copper proteins to bind and contain properly. When the amount of copper in the body, the so-called “free” or “unbound” or “loosely bound” type, becomes excessive, it accumulates in the body and passes through the blood brain barrier, Enter the central nervous system. Since this copper is toxic, over time, it damages and destroys organs such as the brain and liver.

<Treatment of Wilson disease>
When the copper protein network becomes abnormal due to genetic abnormality or aging, copper in the body cannot be properly bound and sequestered by the copper protein and cannot be properly maintained. When copper is present in the so-called “free” or “unbound” or “weakly bound” form, toxicity and oxidative capacity are most prominent. Such free copper is harmful to various organs (eg, liver, brain, etc.). A typical example of such a disease is Wilson's disease. Treatment varies depending on the initial condition of the patient, but is generally treated with a strong copper chelator and complexing agent based on emergency or induction, and free release available from the body for further damage Remove copper or make it unavailable. Copper chelating agents and complexing agents include tetrathiomolybdate, penicillamine or trientine. After the initial emergency or guided therapy, the patient is switched to long-term maintenance therapy, generally over the remaining life. Formulations commonly used for long-term maintenance therapy are those that maintain the poor absorption of copper, such as zinc acetate [Brewer]. Zinc acetate is sold as Galzin ™ in the United States and Wilzin ™ in Europe and is available by prescription. In the United States, other zinc salts available without a prescription are used for long-term maintenance therapy for Wilson's disease patients, but with varying degrees of success. Examples of such other zinc salts include, but are not limited to, zinc carbonate, zinc sulfate, zinc gluconate, zinc oxide, zinc chloride and zinc stearate.

  Zinc is used for comprehensive treatment including initial treatment of Wilson disease [Hoogenraad et al., Lancet, 2: 1262-1263 (1978); Hoogenraad et al., Eur. Neurol., 18: 205-211 (1979); and Hoogenraad et al., J. Neurol. ScL, 77: 137-146 (1987)]. However, zinc is not ideal for initial therapy because of its rather slow action. Thus, it takes about 2 weeks to achieve intestinal metallothionein induction and negative copper balance in patients with Wilson disease [Yuzbasyan-Gurkan et al., J. Lab. Clin. Med., 120: 380-386 [1992] ]. At 2 weeks, as an average average of these patients, zinc reverses copper balance by +0.54 mg daily. However, the induced negative copper balance is fairly mild, averaging -0.35 mg daily [GJ Brewer et al., J. Trace Elem. Exp. Med., 3: 227-234 [1990]; GJ Brewer et al., Amer. J. Med. ScL, 305: (4) 199-202 [1993]]. With zinc treatment, the removal rate of copper is low, so urine copper and non-ceruloplasmin plasma copper (potentially toxic copper as measured in the blood) can be reduced to below toxic levels for as long as 6 months. Cost. As a copper absorption blocker, tetrathiomolybdate (TM) is more effective than zinc. This is because zinc acts only in the region of the small intestine where metallothionein is induced, whereas TM acts all over the gastrointestinal tract. Another advantage of TM is that it works immediately compared to zinc. In addition, there is no lag period necessary for the induction of metallothionein.

  After all, in such long-term maintenance treatment, it is difficult to predict the effect and to set an appropriate dosing schedule because the use of the preparation involves chronic and severe inflammation and nausea in the stomach and esophagus. There is also a need to constantly monitor the value of free serum copper to ensure that free serum copper is maintained within the normal range. For this reason, some patients do not work well. The effects of such formulations vary with the timing of administration because they are related to the timing of the meal. In addition, when the daily prescription is a multidrug administration, it is difficult to comply with the medication, which may cause stomach irritation, and each administration time is performed at least 1 hour before and 3 hours after the meal. There is a need. This compliance also needs to be performed over the entire remaining life of the patient.

<Other disorders associated with increased free copper and / or copper accumulation levels>
Other disorders related to weakly bound copper, increased free copper levels and / or copper accumulation include headache, hypoglycemia, increased heart rate, nausea, anemia, hair loss, nephritis, autism, depression, hallucinations , Hypersensitivity, insomnia, sensory disturbance, paranoia, personality change, psychosis, schizophrenia, mild cognitive impairment, reality escape, atherosclerosis, stroke, tauapathies, synucleinopathies, non-alcoholic fat Hepatitis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, dementia, ALS and autism can be exemplified. Diseases associated with increased inflammation and fibrosis are also associated with increased free copper and can be alleviated by lowering copper levels with copper lowering agents.

<Alzheimer's disease>
The life span of humans has increased significantly since the last century. Alzheimer's disease and other neurodegenerative diseases are increasing in proportion to the population and their impact on life quality is increasing. For example, Alzheimer's disease is a leading cause of dementia in the elderly, accounting for 5-10% of the population over the age of 65 [A Guide to Understanding Alzheimer's disease and Related Disorders, edited by Jorm, New York University Press, See New York (1987)]. Alzheimer's disease shows few signs of memory loss and dementia progresses slowly over the years. The prevalence of Alzheimer's disease and other dementias doubles every five years after age 65 [see 1997 Progress Report on Alzheimer's disease, National Institute on Aging / National Institute of Health]. Alzheimer's disease currently affects 12 million people worldwide, with an estimated 22 million in 2025 and 45 million in 2050 [Alzheimer's See Association Press Release, JuI. 18, 2000].

  The complexity of the brain structure and chemistry, as well as the complexity of brain neurodegenerative diseases (especially Alzheimer's disease), hinders the development of models that exhibit symptoms similar to the changes seen in the human brain. Such models are needed to identify drugs or other formulations that are useful in treating and preventing such diseases and negating the effects of the disease.

  Alzheimer's disease is characterized by the loss of a specific group of neurons and the appearance of two main lesions in the brain called senile plaques and neurofibrillary tangles [Brion et al., J Neurochem. 60: 1372-1382 (1993)]. Elderly plaques occur in the extracellular space. The main element of senile plaques is beta amyloid (A-beta), a naturally secreted but insoluble peptide produced by cleavage of amyloid precursor protein (APP). A-beta is a fragment near the carboxyl terminus of APP.

  Neurofibrillary tangles are filamentous substances that accumulate in loops within neurons. They are most abundant in parts of the brain associated with memory function, such as the temporal hippocampus and its adjoining [Robbins Pathologic Basis of Disease, Cotran et al., [Omicron] .sup.th ed. (See 1999)]. Neurofibrillary tangles are generally present in cortical neurons, particularly the entorhinal cortex, but are also present in hippocampal pyramidal cells, amygdala, brain base and raphe nuclei.

  Neurofibrillary tangles are also seen when the brain is normal, but patients with Alzheimer's disease and other neurodegenerative diseases (progressive supranuclear paralysis, posterior intracranial Parkinson's disease, Pick's disease, amyotrophic lateral sclerosis Is present in the brain of a patient with a disease [Robbins Pathologic Basis of Disease, Cotran et al., 6th ed. (1999), p. 1330]. In previous studies, neurofibrillary tangles are notably associated with, among other things, the decline in cognitive ability associated with disease, as well as directly with neuronal cell death.

  Ultrastructurally, neurofibrillary tangles are composed primarily of helical fiber pairs ("PHF"). The main element of PHF is an abnormally phosphorylated form of protein called tau and its fragments [Robbins Pathologic Basis of Disease, Cotran et al., 6th ed., WB Saunders Company (1999), p.1300 ].

  Tau protein (also called native tau "native tau") is a microtubule-associated phosphoprotein that has been reported to stabilize the cytoskeleton and be involved in determining neuronal shape [Kosik & See Caceres, Cell Sci. Suppl. 14: 69-74 (1991)]. The apparent molecular weight of tau is 55 kDa. Protease cathepsin D cleaves tau protein at neutral (cytoplasmic) pH, resulting in a tau fragment, one of which has a molecular weight of about 29 kDa (some authors refer to as “tau fragment”) [eg, Bednarski & Lynch, J. Neurochem. 67: 1846-1855 (1996); Bednarski & Lynch, NeuroReport 9: 2089-2094 (1998)]. Tau protein and the 29 kDa tau fragment can be phosphorylated. In the normal brain, tau proteins and tau fragments are typically present in an unphosphorylated or dephosphorylated state. However, in neurofibrillary tangles, both tau protein and tau fragments may exist in an abnormally phosphorylated or hyperphosphorylated state. The 29 kDa tau fragment is a major component of neurofibrillary tangles. Hyperphosphorylation impairs the ability of tau proteins to interact with microtubules.

  It has been reported that hippocampal slices were cultured with an inhibitor of cathepsins B and L (N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD)) [Bednarski E, and Lynch G, J Neurochem 67: 1846-55 (1996)]. According to the author's report, this resulted in degradation of the high molecular weight isoform of tau protein, producing a 29-kDa tau fragment (tau 29).

  It has been reported that when hippocampal slices cultured in chloroquine or ZPAD were cultured, the enzymatically active cathepsin D increased and a 29 kDa fragment of tau protein appeared later [Bednarski E, and Lynch G, Neuroreport 9: 2089-2094 (1998)]. The authors state that inactivation of cathepsin L leads to the induction of cathepsin D, which leads to abnormal tau proteolysis, and that such a pathway is likely to play an important role in brain aging.

  In addition to the accumulation of A-beta and neurofibrillary tangles, increased evidence points to a link between lipid metabolism and Alzheimer's disease. Epidemiological studies have shown that patients with increased plasma have reduced lipoprotein cholesterol density and cholesterol levels, and cardiovascular disease has an increased risk of Alzheimer's disease [Kuo, YM, et al , Biochem. Biophys. Res. Comm. 252: 711-715 (1998); Jick, H., et al., Lancet 356: 627-631 (2000)]. Long-term treatment with 3-hydroxy-3-methylglutaryl coenzyme has also been reported, and reductase inhibitors have been reported to reduce the incidence of Alzheimer's disease [Jick, H., et al., Lancet 356: 627-631 (2000); Wolozin, B., et al., Arch. Neurol. 57: 1439-1443 (2000)].

  In vitro experiments have been performed in conjunction with links to lipid metabolism, and cholesterol has been shown to affect beta amyloid (A-beta) production and aggregation [Bodovitz, S., and Klein, WL, J Biol. Chem. 271: 4436-4440 (1996); Xu, H., et al., Proc. Natl. Acad. Sci. USA 94: 3748-3752 (1997); Howland, DS, et al., J Biol. Chem. 273: 16576-16582 (1998)]. Transgenic mice fed a diet high in cholesterol have also been reported to have increased A-beta deposits [Refolo, LM, et al., Neurobiol. Dis. 7: 321-331 (2000) ].

  ApoB and apoE mediated transport of cholesterol to lysosomes is an important step for cells to utilize these sterols and is particularly important for mature neurons that rely on extracellular apoE mediated transport of cholesterol [Brown, MS, and Goldstein, JL, Annu. Rev. Biochem. 52: 223-261 (1983)]. Within lysosomes, lysosomes and other lipids are separated from ApoE [Brown, MS, and Goldstein, JL, Annu. Rev. Biochem. 52: 223-261 (1983)].

  Changes in cholesterol levels are associated with some neurodegenerative diseases. For example, insoluble A-beta 1-42 is present in Neiman-Pick C (NPC) mutant cells [Yamazaki, T., et al., J. Biol. Chem. (2000)]. These adoptions exhibit many pathological features, one of which is an impairment of intracellular transport of cholesterol [Millard, EE, et al., J. Biol. Chem. 275: 38445-38451 (2000 )]. ApoE4 isoforms are also known to be risk factors for late-onset Alzheimer's disease.

  Inhibition of cholesterol synthesis promotes tau phosphorylation in isolated cell cultures [Sawamura, N., et al., J. Biol. Chem. 57: 1439-1443 (2001))] . Similarly, tau hyperphosphorylation was observed in cell cultures prepared from NPC mutant mice [Sawamura, N., et al., J. Biol. Chem. 57: 1439-1443 (2001). ]. Gradually progressive disorders in lysosomes affect the sorting / trafficking of cholesterol from lysosomes and late endosomes, causing pathologies for neurodegenerative diseases and Alzheimer's disease.

  U.S. Patent No. 6,803,233 describes an animal model of Alzheimer's disease, where cysteine protease inhibitors can create an animal model of Alzheimer's disease that includes Hallmark neurofibrils (NFTs) that concentrate tau helical filament pairs. It describes what you can do. However, this patent does not describe the copper-binding effect of cysteine, and for low molecular weight copper-cysteine complexes that can cross the blood brain barrier and up-regulate the production of APP, Aβ and tau proteins, It is not described that homocysteine makes its available pool. The present invention, in one aspect, relates to a preparation of zinc (more preferably, gastroretentive sustained release zinc) and folic acid, wherein the systemic and low molecular weight copper cysteine complexes (eg, copper-homocysteine) It lowers CSF levels and stabilizes them. The inventor recognizes that these will contribute to neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and ALS.

<Prior art for copper involvement in Alzheimer's disease, Parkinson's disease, ALS and other central nervous system (CNS) diseases>
Many conflicting conclusions and hypotheses have been reported so far regarding the regulatory role of elemental copper and zinc in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, ALS and CJD. For example, many of the hallmark proteins associated with Alzheimer's disease are known to be copper-binding proteins, such as amyloid precursor protein (APP), beta amyloid (Aβ) (peptides 1-40 and 1-42). ), Tau (helical fragment pairs (PHFs) and neurofibrillary tangles (NFTs)), beta secretase (BACEl) and apolipoprotein E (apoE) (3 major human variants: apoE2, apoE3 and apoE4) There is. For the latter, apoE2, apoE3 and apoE4 differ only at positions 112 and 158 with or without a cysteine residue. It is known that apoE2, apoE3 and apoE4 have different copper binding capacities.

  Epidemiological genetic studies have shown that apoE4 mutants without cysteine residues at positions 112 and 158 increase the risk of AD, whereas apoE2 with two cysteine residues at positions 112 and 158 is a human mutation Compared to the body's most common apoE3 (which has only cysteine residues at these positions), it is believed that it will benefit from gaining protection against Alzheimer's disease (as well as athlerosclerosis).

  A study by Framingham reports that increased homocysteine levels are associated with an increased risk of Alzheimer's disease, but homocysteine is a low molecular weight copper-binding protein that is toxic and weakly bound. It is not described that sex replaceable “free” copper is delivered to, maintained in, and slowly eliminated from the CNS.

  An increase in cholesterol levels is also associated with an increased risk of Alzheimer's disease. In particular, the oxidized cholesterol 27S-hydroxy-cholesterol and / or 24S-hydroxy-cholesterol are both present in the CNS and circulating and Alzheimer's patients (as well as athlerosclerosis). In addition to Alzheimer's disease, elemental copper is a hypothesis that superoxide dismutase (SODl), a mutant of copper / zinc binding protein, plays a role in reducing copper-binding ability in other neurodegenerative diseases such as ALS. It has been built.

  In Parkinson's disease, the protein alpha-synuclein (AS), which binds copper, iron and aluminum, is known to be a major component of neuronal and glial cytoplasmic inclusions (also known as Lewy Bodies). It has been. This inclusion body is widely considered to be a hallmark lesion of both Parkinson's disease and a neurodegenerative disease called synucleinosis.

<Defect of neural tube>
Neural tube defects (NTDs) are the primary congenital defects in the fetal brain or spine that occur when the neural tube (which later becomes the brain and spine) does not form normally and damages the brain or spine. This happens within 2-3 weeks of pregnancy and may be unaware of pregnancy. It has been reported that the incidence of NTDs is reduced by up to 70% if the mother properly takes B vitamins and folic acid before pregnancy, but how folic acid works has not been described so far [CDC, Folic Acid Now, CDC-NCEH99-0463, Nov. 2005].

  Spina bifida and anencephalopathy are two common NTDs. In the United States, approximately 3,000 pregnant women are affected by spina bifida and anencephalopathy each year. Vertebral tears occur when the spine and spine do not close all the time. When this happens, the spinal cord and spine are not formed in their original state. The fluid sac passes through the baby's back opening. Much of that time, parts of the spinal cord are in this sac and are damaged. Many children born with spinal vertebrae can complete their lives, but can also live their lives as disabled people and require many surgical operations.

  Not all children born with spinal vertebrae have the same requirements. Some children's problems are more severe. But even in that case, with the right treatment, many children will be able to grow and live a full and productive life.

  Anencephalopathy occurs when the brain and skull are not formed correctly. When this happens, some or all of the brain and skull are missing. A baby with this defect either dies before being born (abortion) or immediately after birth.

  Folic acid is useful in preventing other birth defects such as cleft lip and palate and some heart defects. Ingestion of folic acid also has other health benefits for both men and women. Mothers of children with cleft lip and palate have been reported to be low in zinc and high in copper [Hoyasz KK, Wiad. Lek., 58 (7-8): 382-5 (2005)]. To date, nothing has been described about the role of folic acid in reducing the pool of circulating serum copper bound to homocysteine.

<Antioxidant>
In another aspect of the present invention, a formulation useful for reducing and maintaining systemic and CSF levels of free copper and iron in a stable manner is provided, the formulation using one or more antioxidants. As prepared and oxidizing agents, for example, vitamin C, vitamin E, QlO, Onega 3 fatty acid, zinc cysteine or a complex thereof can be mentioned.

<Acetylcholinesterase inhibitor>
Acetylcholinesterase inhibitors are highly evaluated as clinical preparations for treating and improving senile dementia (eg, Alzheimer-type senile dementia), cerebrovascular dementia, attention deficit hyperactivity disorder and schizophrenia. In particular, donepezil hydrochloride (l-benzyl l-4-[(5,6-dimethoxy-l-indanone) -2-yl] methylpiperidine hydrochloride) is an acetylcholinesterase that produces the desired pharmacological activity with few side effects. It has been reported. As acetylcholinesterase inhibitors, in addition to donepezil hydrochloride, rivastigmine (3- [l- (dimethylamino) ethyl] phenyl N-ethyl-N-methylcarbamate), merifonate (dimethyl 2,2,2-trichloro-1- Hydroxyethyl) phosphate), tacrine hydrochloride (1,2,3,4-tetrahydro-9-acridinamine), galantamine hydrobromide, neostigmine, physostigmine and the like. The object of the present invention is to remove acetylcholinesterase inhibitors from zinc, zinc-cysteine, tetrathiomolybdate, gastroretentive sustained release zinc preparations and other controlled release preparations of essential trace metals (copper, iron, etc.). It is blended with a preparation selected from the group consisting of:

<NMDA receptor antagonist>
Overactivity of ionotropic glutamate receptors sensitive to N-methyl-D-aspartate (NMDA receptor) is known to lead to neuronal death and mediate various neurological diseases [Choi, Neuron 1: 623-634 (1988)]. Glutamate, an excitatory neurotransmitter, can cause hypoxic ischemic damage when accumulated in the brain in large quantities, which is an ionotropic glutamate receptor that can penetrate Ca ²⁺ and Na ⁺ The body is activated to cause neuronal death [Choi and Rothman, Annu Rev Neurosci 13: 171-182 (1990)]. Antagonists of NMDA receptors significantly reduce brain damage after hypoglycemia, hypoxia or hypoxic ischemia [Simon, Swan, Griffiths, and Meldrum. Science 226: 850-852 (1984); Park, Nehls, Graham, Teasdale, and McCulloch, Ann Neurol 24: 543-551 (1988) .; Wieloch, Science 230: 681-683 (1985); Kass, Chambers, and Cottrell, Exp. Neurol. 103: 116-122 (1989) Weiss, Goldberg, and Choi, Brain Res. 380: 186-190 (1986)]. Thus, NMDA receptor antagonists have therapeutic potential that can protect the brain from brain damage due to hypoglycemia, hypoxia or hypoxic ischemia.

  Excitotoxicity is thought to be involved in neurodegeneration due to traumatic brain injury (TBI). The value of quinolinic acid, an endogenous agonist of NMDA receptors, increased 5 to 50-fold in human patients with TBI [EH Sinz, PM Kochanek, MP Heyes, SR Wisniewski, MJ Bell, RS Clark, ST DeKosky, AR Blight, and DW Marion]. Quinolic acid is also increased in cerebrospinal fluid and has been reported to be involved in human post-TBI mortality [J. Cereb. Blood Flow Metab. 18: 610-615, (1998)]. In animal models of brain injury, it has been reported that glutamate and aspartate levels are significantly increased [Faden, Demediuk, Panter, and Vink, Science 244: 798-800 (1989)]. Glutamate release was also observed in the spinal cord after shock injury in rats [Demediuk, Daly, and Faden. J Neurochem J. Neurochem. 52: 1529-1536 (1989)]. Antagonists of NMDA receptors reduce neuronal death after traumatic brain injury or spinal cord injury [Faden, Lemke, Simon, and Noble. J. Neurotrauma. 5: 33-45 (1988); Okiyama, Smith, White, Richter, and Mclntosh. J. Neurotrauma. 14: 211-222 (1997)].

  Glutamate plays a central role in the induction and propagation of convulsions [Dingledine, McBain, and McNamara, Trends. Pharmacol. ScL 11: 334-338 (1990); Holmes. Cleve. Clin. J. Med. 62: 240 -247 (1995)]. NMDA receptor antagonists have been reported to act as anticonvulsants and antiepileptics in various models of epilepsy [Anderson, Swartzwelder, and Wilson, J. Neurophysiol. 57: 1-21 (1987); Wong, Coulter, Choi, and Prince. Neurosci. Lett. 85: 261-266 (1988); McNamara, Russell, Rigsbee, and Bonhaus, Neuropharmacology 27: 563-568 (1988)].

  Amyotrophic lateral sclerosis (ALS) is accompanied by degeneration of both upper and lower motor neurons and exhibits symptoms of neurogenic atrophy, muscle weakness and fiber bundle contraction. The etiology of ALS has not been elucidated, but excitotoxicity is expected to be involved in the ALS process. In particular, ALS patients have been shown to have increased levels of extracellular glutamate and impaired glutamate transport. When neurostimulators were administered, the spinal cord of ALS patients showed pathological changes [Rothstein. Clin. Neurosci. 3: 348-359 (1995); Ikonomidou, Qin, Labruyere, and Olney J. Neuropathol. Exp. Neurol. 55 : 211-224 (1996)].

  Antagonizing the NMDA receptor may be applicable to the treatment of Parkinson's disease (PD). Several antagonists of NMDA receptors protect dopaminergic neurons from the neurotoxin MPTP (l-methyl-4-phenyl-l, 2,3,6-tetrahydropyridine) [Lange, Loschmann, Sofic, Burg , Horowski, Kalveram, Wachtel, and Riederer. Naunyn Schmiedebergs Arch. Pharmacol. 348: 586-592 (1993); Brouillet and Beal. Neuroreport. 4: 387-390 (1993)]. NMDA receptor antagonists can also improve levodopa-induced dyskinesia and improve the therapeutic effects of levodopa [Papa and Chase, Ann. Neurol. 39: 574-578 (1996); Marin, Papa Engber, Bonastre, Tolosa, and Chase, Brain Res. 736: 202-205 (1996)]. Two NMDA receptor antagonists, memantine and dextromethophane, have been reported to be useful in the treatment of PD patients [Verhagen, Del Dotto, Natte, van den Munckhof, and Chase, Neurology 51: 203 -206 (1998); Merello, Nouzeilles, Cammarota, and Leiguarda. Clin. Neuropharmacol. 22: 273-276 (1999)].

  Huntington's disease (HD) is a progressive neurodegenerative disease that acts primarily on small and medium sized interneurons, but sparing NADPH-diaphorase neurons containing somatostatin and neuropeptides in the striatum ( sparing). These pathological features of HD are observed in striatal tissues following intrastriatal injection of quinolic acid or cultured striatal neurons exposed to NMDA, and NMDA receptor-mediated neurotoxicity is a selective neuron in HD Increases the possibility of death [Koh, Peters, and Choi, Science 234: 73-76 (1986); Beal, Kowall, Ellison, Mazurek, Swartz, and Martin, Nature 321: 168-171 (1986 ); Beal, Ferrante, Swartz, and Kowall. J. Neurosci. 11: 1649-1659 (1991)].

  Therefore, other objects of the present invention include NMDA antagonists (e.g., memantine, flupirtine, etc.), zinc, zinc-cysteine, tetrathiomolybdate, gastroretentive sustained release zinc preparations and other essential trace elements ( Preparations formulated with a formulation selected from the group consisting of sustained release preparations of copper, iron, etc.).

<Free radicals and brain diseases>
Free radicals are produced by alterations in brain regions following hypoxic ischemia or traumatic brain and spinal cord injury [Hall and Braughler, Free Radic. Biol. Med. 6: 303-313 (1989); Anderson and Hall Ann. Emerg. Med. 22: 987-992 (1993); Siesjo and Siesjo, Eur. J. Anaesthesiol. 13: 247-268 (1996); Love, Brain Pathol. 9: 119-131 (1999)]. Free radicals scavenging antioxidants or maneuvers reduce brain damage due to hypoxic-ischemic or traumatic injury [Faden, Pharmacol. Toxicol. 78: 12-17 (1996); Zeidman, Ling, Ducker, and Ellenbogen, J. Spinal. Disord. 9: 367-380 (1996); Chan, Stroke 27: 1124-1129 (1996); Hall, Neurosurg. Clin. N. Am. 8: 195-206 (1997)]. Free radicals are brain regions that undergo alterations in neurodegenerative diseases due to point mutations in Cu / Zn superoxide dismutase in ALS, increased glutathione and decreased iron in PD, iron accumulation in AD, or mitochondrial dysfunction in HD. There is much evidence that it is generated [Rosen, Siddique, Patterson, Figlewicz, Sapp, Hentati, Donaldson, Goto, O'Regan, and Deng. Nature 362: 59-62 (1993); Jenner and Olanow, Neurology 47: S161-S170 (1996); Smith, Harris, Sayre, and Perry, Proc. Natl. Acad. Sci. USA 94: 9866-9868 (1997); Browne, Ferrante, and Beal, Brain Pathol. 9: 147-163 ( 1999)]. Therefore, antioxidants were neuroprotective against such neurodegenerative diseases [Jenner, Pathol. Biol. (Paris.) 44: 57-64 (1996); Beal, Ann. Neurol. 38: 357 -366 (1995); Prasad, Cole, and Kumar.J.Am.Coll.Nutr. 18: 413-423 (1999); Eisen and Weber, Drugs Aging 14: 173-196 (1999); Grundman, Am.J .Clin.Nutr. 71: 630S.-636S (2000)].

<Zinc and brain disease>
Zn ²⁺ mediates the neurodegenerative processes observed in epilepsy, ischemic disease, trauma and Alzheimer's disease (AD). The pharmacological activity of the kainate receptor by administration of the kainic acid epilepsy-inducing excitatory toxin translocates Zn ^{2+ to} post-synaptic degenerative neurons in several forebrain regions [Frederickson, Hernandez, and McGinty. Brain Res. 480: 317-321 (1989)].

In the past, there has been interest in reducing central nervous system zinc using chelating agents and / or blockers. Blockers that translocate Zn ²⁺ with Ca-EDTA reduce neuronal loss after transient forebrain ischemic or traumatic injury [Koh, Suh, Gwag, He, Hsu and Choi, Science 272 : 1013-1016 (1996); Suh, Chen, Motamedi, Bell, Listiak, Pons, Danscher, and Frederickson, Brain Res. 852: 268-273 (2000)]. Zn ²⁺ is observed in extracellular plaques and degenerated neurons in AD and is thought to result in neuronal alterations in AD [Bush, Pettingell, Multhaup, Paradis, Vonsattel, Gusella, Beyreuther, Masters, and Tanzi, Science 265 : 1464-1467 (1994); Suh, Jensen, Jensen, Silva, Kesslak, Danscher, and Frederickson. Brain Res. 852: 274-278 852 (2000)]. This document conducts clinical trials of copper / zinc chelators, quinoform, and PBT-01 for the treatment of Alzheimer's and Huntington's disease. Other aspects of the present invention seem to contradict such proposals, but to increase intestinal, systemic and cerebrospinal fluid (CSF) values for zinc and induce intestinal metallothienein. Including the use of gastroretentive sustained release preparations, which maintain the stable value of weakly bound and free copper in the systemic circulation and CSF.

<Cholesterol>
Sterols are structural lipids that are present in the membranes of all eukaryotic cells. These lipids are rigid and are characterized by a four-ring hydrocarbon steroid nucleus. Sterols are required not only to confer membrane fluidity, but also to act as precursors for various products with specific biological activities. For example, cholesterol is an amphiphilic sterol with a polar hydroxyl head group and a nonpolar hydrocarbon body (steroid nucleus), and is the main sterol present in animal tissues. Cholesterol is an essential molecule that plays an important role in the structural integrity of the cell membrane, becomes a precursor of steroid hormones, and functions as a precursor of bile acids. Cholesterol is synthesized in all organs, but is synthesized from acetate, particularly in the liver, and is obtained through dietary intake.

  Cholesterol is a necessary molecule, but high levels of blood cholesterol carried in apoB-containing lipoproteins or hypersterolemia are involved in atherosclerosis, heart attacks and strokes [Schultheis, 1990; Mitchell, 1990)]. Hypersterolemia, if not controlled, is one of several conditions that lead to coronary artery disease. Coronary artery disease is a leading cause of death in the United States, with a number of approximately 600,000 people annually. Therefore, there is a need for therapeutic methods that can lower cholesterol levels and methods for screening patients at risk for high cholesterol.

  Possible therapeutic targets are transcriptional regulators involved in cholesterol metabolism. One such factor, the nuclear receptor, is a ligand-activated transcriptional regulator that governs aspects of all major developmental and metabolic pathways [reviewed in Kastner et al., 1995; Mangelsdorf et al. , 1995]. For example, LXRs were first identified as “orphan” members of the nuclear receptor superfamily of unknown ligand and function [Willy and Mangelsdorf, 1998]. LXRs have recently been activated by naturally occurring oxidized derivatives of certain classes of cholesterol (e.g. 22 (R) -hydroxycholesterol, 24 (S) -hydroxycholesterol and 24,25 (S) -epoxycholesterol) [Janowski et al., 1996; Lehmann et al., 1997]. Oxysterols are concentrated in tissues with high cholesterol metabolism and high LXR expression (eg, liver, brain and placenta) [Lavy et al., 1977; Spencer et al., 1985; Lutjohann et al., 1996].

  LXRs function as heterodimers with retinoid X receptors (RXRs), and RXR / LXR complexes can be activated by RXR ligands (i.e. rexinoids) and oxysterols [Teboul et al., 1995; Willy et al., 1995; Janowski et al., 1996). Two types of LXR proteins (alpha and beta) are known to exist in mammals. LXRalpha expression is limited and is highest in the liver (hence referred to as liver X receptor) and low but significant in the kidney, intestine, spleen and adrenal gland [Apfel et al., 1994; Willy et al., 1995]. LXR.beta. Has been reported to be more extensive and present in almost all tissues examined [Shinar et al., 1994; Song et al., 1994].

  The pattern of expression of LXRs and their oxidized sterol ligands suggests that these receptors may have a role in cholesterol metabolism. Cholesterol has three essential metabolites in mammals: esterification (for transport or storage) and conversion to steroid hormones or bile acids. Steroid hormone synthesis is known to be governed by the orphan nuclear receptor steroidogenic factor-1 (SF-I) [Parker and Schimmer, 1997]. It is possible that LXRs are involved in the synthesis of bile acids [Janowski et al., 1996]. A promising target for any bile acid derivative is cholesterol 7.alpha.-hydroxylase (Cyp7a), the rate-limiting enzyme in the classical bile acid synthesis pathway [Janowski et al., 1996; Lehmann et at., 1997]. The Cyp7a promoter contains a functional LXR responsive element that can be activated by RXR / LXR heterodimers as oxysterol-dependent and retinoid-dependent [Lehmann et (al. , 1997)]. Bile acid production is one of two major pathways of cholesterol catabolism and excretion in mammals [Russell and Setchell, 1992]. Perturbations in this pathway cause a variety of diseases including cholesterol gallstones, atherosclerosis and Alzheimer's disease. These observations raised the interesting possibility that LXRs function as transcriptional control points in bile acid metabolism.

  RXR homodimeric and heterodimeric RXR proteins have been observed to be regulated by 9-cis retinoic acid that ties to the carboxyl terminal of RXR [Mangelsdorf and Evans, 1995]. RXR can generate heterodimers with many other proteins in the nuclear receptor superfamily (including LXR). Depending on the receptor protein that dimerizes with RXR and the ligand present, the effect of the heterodimer on transcription varies. Synthetic retinoids have been reported to selectively bind and activate RXRs [US Pat. Nos. 5,780,676 and 5,455,265]. US Pat. No. 6,835,866 describes the use of RXR specific ligands (eg LG100268) to improve liver clearance of cholesterol.

  The ability to modulate lipid concentrations in vivo by targeting nuclear hormone receptor superfamily proteins with specific ligands may be useful in the treatment of various diseases related to lipid metabolism. For example, high blood cholesterol levels are associated with coronary artery disease. Reducing cholesterol intake in the diet can significantly reduce cholesterol levels for many people. However, reducing dietary intake of cholesterol may not be sufficient for those with high blood cholesterol levels due to inefficient endogenous cholesterol homeostasis. For these people, the ability to lower blood cholesterol levels will prove very beneficial. Currently, there are various drugs for treating high cholesterol blood levels and other abnormal blood lipid levels. For example, cholestyramine and colestipol are resins that bind bile acids in the intestine and lower cholesterol by converting cholesterol to bile acids. In order to reduce the blood lipid concentration, statins (eg, atorvastatin, lovastatin, pravastatin, etc.) are generally used in addition to nicotinic acid, gemfibrozil, and probucol.

  In order to improve bile duct function, bile acids such as ursodeoxycholic acid (UDCA) 17.beta .- (l-methyl-S-carboxypropyl) ethiocholane-3.alpha., 7beta.diol are also known. It has been. The main toxicities associated with UDCA are unknown [W. H. Bachrach et al., Dig. Dis. ScL, 30, 642 (1985)]. As pharmaceutically acceptable salts and esters of UDCA, (C1-C4) alkanoic acid (non-toxic ester with alkanoic acid (such as formic acid, acetic acid or propionic acid)) of free hydroxy group of UDCA, phosphate ester of OH group, free ( Mention may be made of (C24) carboxylic acid groups and (C1-C4) alkyl esters of non-toxic alkali metals, ammonia salts or amine salts of free carboxylic acid moieties. The esters and salts can be readily prepared from free UCDA by methods well known in the art. At least diformate and diacetate are known compounds [see The Merck Index (1lth ed. 1989) at 1556]. UDCA is commercially available as a 300 mg hard gelatin capsule under the trade name ACTIGALL (Summit Pharmaceuticals, Summit, NJ). This is prescribed for the dissolution of gallstones.

  One aspect of the present invention is that bolus administration of soluble copper and iron contained in tap water occurs because weakly bound or so-called free copper and iron in the systemic circulation and CSF undergo peak fluctuations in one day. And is based at least in part on the unique finding that it is exacerbated by dissolution of the initially released copper or iron supplement. Furthermore, as is common in older adults, in those with reduced liver or biliary dysfunction, free copper levels in the systemic circulation and CSF are maintained at high levels for long periods of time due to impaired liver clearance. Therefore, it is particularly susceptible to neurodegenerative diseases related to copper and iron homeostasis in CNS such as Alzheimer's disease, Parkinson's disease and ALS. Therefore, further features of the invention described below are formulations that can improve liver and bile clearance function (statins (including atorvastatin), RXR specific ligands (such as LG1002688)), zinc, zinc- It is formulated with a preparation selected from the group consisting of cysteine, tetrathiomolybdate, gastroretentive sustained release zinc preparations and other controlled release preparations of essential trace metals (copper, iron, etc.), liver and Can improve hepatic incorporation and clearance of free copper and iron in the systemic circulation and CSF of people with impaired bile duct function, such as Alzheimer's disease, mild cognitive impairment, Parkinson's disease, ALS and atherosclerosis In addition, it can treat or prevent neurodegenerative disorders caused by increased and variable levels of free copper and iron in the systemic circulation and CSFPreferentially, such preparations reduce the potential for hypocopia and anemia through the administration of chroic zinc. Such preparations are preferentially bound to copper, iron and / or certain copper and iron binding excipients (such as howay, plant fiber, metallothionein, dry milk or infant formula). Added to sustained release formulations containing other essential trace elements.

<Parkinson's disease>
Parkinson's disease (PD) is a well-known neurodegenerative disease, first published in 1817 by James Parkinson. There are four main diagnostic signs of illness: resting tremor, slow motion, muscle rigidity and postural reflex disorder. These signs of motor dysfunction are caused by the loss of dopaminergic neurons in the heterogeneous linear system [Gibb, W., et al., J. Neurol. Neurosurg. And Psych., 51: 745-52 (1988 )].

  PD is characterized by Lewy body production and death of dopaminergic neurons [Adams D. et al., Principles of Neurology, 874-880, 3rd Edition, McGraw-Hill, N.Y., (1985)]. The neuropathological hallmark of PD is a Lewy body. Lewy bodies are intracytoplasmic inclusions in degenerative neurons consisting of a dense core of filamentous and granular material surrounded by radically oriented filaments 10-20 nm in diameter [Goedert, 20 M., et al., Curr. Op. Neurobio. 8: 619-32 (1999)]. In general, the cause of PD is unknown, and there has been an active debate about the relative role of genetic and environmental factors [Tanner, C, et al., JAMA, 281: 341-6 (1999)]. Manganese exposure has induced parkinsonism, including schizophrenia, in miners. As an example of epidemiological studies, the relationship between industrial exposure to iron and the occurrence of PD [Corell J. M et al., Toxicol. Appl. Pharmacol., 80: 467-72, (1985)] Relationship with mercury concentration in blood [Ngim CH et al., Neuroepi., 8 (3): 128-141 (1989)] and with death rates from PD and proximity to iron-related industrial processes [Rybicki A. et al ., Mov Disord., .8 (1): 87-92_ (1993)].

  Alpha-synuclein was originally identified as a protein that is up-regulated in relation to elongation during the critical period of zebra finch's twitter learning ability [George M., et al., Neuron, 15: 361 (1995). )]. Alpha-synuclein is a ubiquitous protein that shares significant physical and functional homology to the protein chaperone 14-3-3, and is particularly abundant in the brain, Ostrerova N. et al., J. Neurosci. 19: 5782 (1990); Clayton D. et al., TINS 21: 249 (1998)]. Alpha-synuclein is usually phosphorylated at serines 87 and 129 [Okochi M. et al., J. Biol. Chem., 275: 390 (2000)]. Recent studies have reported that alpha-synuclein mutations cause familial PD, and that alpha-synuclein accumulates in Lewy bodies. These findings suggest that alpha-synuclein is involved in the pathophysiology of PD [Spillantini M. et al., Nature, 388: 839 (1997); Spillantini M. et al., PNAS USA, 95 : 6469 (1998); Jenner P. et al., Ann. Neurol., 44: S72 (1998)]. To date, the only identified mutations for familial PD are the A53T and A30P variants of alpha-synuclein [Goedert, M., et al., Curr. Op. Neurobio., 8: 619-32 (1999 Papadimitriou, A., et al., Neurology, 52: 651-4 (1999); Polymeropoulos, M., et al., Science, 276: 1197-9 (1997)]. However, there is a lot of situational evidence related to oxidative stress as the etiology of the disease [Jenner, P., et al., Annual Neurol., 44: S72-84 (1998)].

  There is a variety of experimental evidence suggesting that Lewy bodies interact with alpha-synuclein. For example, immunohistochemistry studies have shown that Lewy bodies stain strongly against alpha-synuclein and ubiquitin [Jenner, P., et al., Annual Neurol., 44: S72 -84_ (1998); Markopoulou, K., et al., Annual. Neurol., 46: 374-81 (1999); Spillantini, M., et al., Nature, 388: 839-40 (1997); and Spillantini, M, et al., Proc. Natl. Acad. Sci. USA, 95: 6469-73 (1998)]. In vitro experiments using recombinant proteins suggest that mutants A53T and A30P have increased alpha-synuclein aggregation compared to wild-type alpha-synuclein [Conway, K., et al ., Nature Med., 4: 1318-20 (1998); Giasson, B., et al., J. Biol. Chem., 274: 7619-22 (1999); Hashimoto, M., et al., Brain Res., 25 799: 301-6 (1998)].

  One of the most important problems with alpha-synuclein aggregation and Lewy body production is whether these processes are harmful to cells. Lewy bodies are either inert tombstone markers that occur in response to free radical damage or are poisonous to cells. Both examples are given in the literature. Aggregated amyloid-beta (beta) is toxic to neurons, but lipfustin appears to be innocuous to cells [Behl, C, et al., Cell 77: 817-27 (1994)]. Huntington's protein provides an intermediate state, and the toxicity associated with Huntington's disease may prevail over aggregation, and even aggregation of Huntington's protein may even have a protective effect [Saudou, F., et al., Cell 95: 55-66 (1998)]. Among previous studies, it has been reported that transient overexpression of alpha-synuclein is toxic to various cells, including two cell lines, SK-N-SH and PC12 [Ostrerova , N., et al., Neurosci., 19: 5782-91 (1999)]. Consistent observations have recently been published, in which alpha-synuclein overexpression in mice has been reported to be toxic with age-related loss of dopaminergic terminals and movement disorders [Masliah, E et al., Science, 287: 1265-1269 (2000)]. These findings suggest that increased alpha-synuclein aggregation rates are involved in the mechanism of neurodegeneration in PD and other Lewy body diseases.

  Recent studies on transgenic animals also suggest that alpha-synuclein aggregation is detrimental to neurons. It has been reported that dopaminergic dysfunction occurred in transgenic mice expressing wild-type human alpha-synuclein [Masliah, E., et at., Science, 287: 1265-1269 (2000)]. Furthermore, Drosophila overexpressing alpha-synuclein has been reported to lead to dopaminergic dysfunction and dopaminergic neuronal death in association with increased alpha-synuclein aggregates [Feany, MB, et al., Nature 404: 394-8 (2000)]. It has been suggested that neurons with dopamine promote alpha-synuclein aggregation and degenerate by increasing these aggregates.

  Oxidative stress caused by iron and hydrogen peroxide has recently been reported to induce amyloid-like aggregate formation of alpha-synuclein in vitro [Hashimoto, M., et at., NeuroReport, 10: 717-21 (1999); Paik, S., et al., Biochem. J., 340: 821-8 (1999)]. Oxidative stress is thought to be involved in PD because dopamine has a strong free radical generating action and is the main neurotransmitter in the substantia nigra [Chiueh, C, et at., Adv. Neurol., 60: 251-8 (1993); Jenner, P. et al., Ann. Neurol., 25 44: S72-84 (1998)]. Iron also promotes free radical production and accumulates in the substantia nigra with age [Jenner, P., et al., Ann. Neurol., 44: S72-84 [1998)]. Iron is deposited as hemosiderin grains in the cytoplasm, and mitochondria filled with ferritin grains have been reported to be observed in the ventral lateral thalamus, caudate and lens nucleus and substantia nigra of Parkinson syndrome [Earle M., J. Neuropathol. Exper. Neurol., 27 [1): 1-14, (1968); Asenjo A. et al., Rev. Neurologique, 121 [6): 581-92, (1969); Riederer P., et al., J. Neurochem., 52 [2): 515-20, [1989)].

<Non-alcoholic steatohepatitis>
Nonalcoholic steatohepatitis (NASH) is associated with the progression of systemic changes in the liver and can be compared to that caused by excessive consumption of alcohol (but not to alcohol dependence). NASH is characterized by Mallory bodies with large and / or droplet properties, lobular and portal vein inflammation, and sometimes fibrosis and cirrhosis. NASH is also commonly associated with hyperlipidemia, obesity and type II diabetes. Other clinical conditions characterized by hepatic steatosis and inflammation include excessive fasting, jejunal ileal bypass, complete parenteral nutrition, chronic hepatitis C, Wilson disease and drugs (corticosteroids, calcium channel blockers, high-dose Side effects of synthetic estrogens, metrexate, amiodarone, etc.). Therefore, the term “nonalcoholic steatohepatitis” refers to patients with these biopsy findings: (a) significant drinking, (b) a previous survey of weight loss, (c) drugs for steatohepatitis History of use, (d) used to describe in relation to evidence of genetic liver disease, or (e) lack of chronic hepatitis C infection [JR Ludwig et al., Mayo Clin. Proc, 55, 434 ( 1980) and EE Powell et al., Hepatol., 11, 74 (1990)]

  The etiology of NASH is unknown. There appears to be a correlation between the degree of steatosis and the degree of fibrosis [see, eg, I. R. Wanless et al., Hepatology, 12, 1106 (1990)]. Increases in free fatty acids in hepatocytes can cause inflammation-related membrane damage, cholestasis, and organelle dysfunction. Cell death and fibrosis persist inflammation, and cirrhosis occurs when wounds persist. Steatohepatitis can be an important cause of end-stage liver disease and may be responsible for an enormous number of cases of clyptogenic cirrhosis [E. E. Powell et al]. Unfortunately, once cirrhosis occurs, the only treatment strategy would be liver transplantation. Therefore, there is a need for an effective treatment for nonalcoholic steatohepatitis.

<Multiple sclerosis>
Elemental copper is also known to play a role in the production of myelin. The antigenic form of myelin is also associated with T cell-mediated lesions and myelin decay that are characterized by multiple sclerosis.

<Autism>
Metabolic copper dysfunction has also been implicated in autism [Chuahan, A et. Al, Life Sci. 2005 Oct 8; 75 (21): 2539-49]. Therefore, another further object of the present invention is to treat autism with gastroretentive and / or sustained release formulations of zinc, copper, iron and other trace metals, Including maintaining a target stable level for free copper and iron in the CSF.

  One aspect of the present invention is to induce a state of inhibition of copper absorption over a long period of time in a human or animal in need of reducing, managing or maintaining free, unbound or weakly bound copper to a low level. And provides improved pharmaceutical compositions, kits and methods as a means of monitoring, maintaining safety, and not as a symptomatic disease, including but not limited to Wilson's disease, Alzheimer's disease, atherosclerotic arteries In addition to sclerosis, autoimmune diseases, oxidative stress, old age-related copper excretion disorders, liver diseases, neurodegenerative diseases such as ALS, Parkinson's disease, multiple sclerosis, as well as increased copper protein levels like schizophrenia List relevant illnesses. Also disclosed are specially prepared pharmacants, kits, and dosing regimes that are intended to reduce the need to monitor patients with signs of hypercopperemia.

  Thus, one aspect of the present invention is an over-the-counter prodocuts designed and prepared to inhibit the absorption of copper and copper ions from the gastrointestinal tract and to limit systemic bioavailability. )I will provide a.

<A novel copper-containing formulation that induces the target stable copper balance>
Zinc acetate developed as immediate-release 25 mg and 50 mg capsules was prepared and clinical trials were conducted as maintenance therapy for the treatment of Wilson disease. On January 28, 1997, the FDA announced that 25 mg of zinc acetate mixed with corn starch and magnesium stearate in immediate-release gelatin capsules for the continuous treatment of patients with Wilson disease who were initially treated with a copper chelator. And approved for 50 mg formulation. The recommended dose for Wilson's disease patients is 50 mg three times a day (tid), at least 1 hour before eating and drinking (excluding water) and at least 1 hour after eating and drinking (excluding water).

Zinc inhibits intestinal absorption of copper from the diet and resorption of endogenously secreted copper such as copper from saliva, gastric juice and bile. Zinc induces the production of metallothionein and protein that binds copper in intestinal cells, thereby preventing the capsule from entering the blood. Bound copper is lost in the stool after enteric cell desquamation. Aside from meals and beverages, Wilson's disease patients who took 50 mg of immediate-release zinc acetate capsule three times a day showed a negative average copper balance of -0.44 mg / day with a suitable average Cu ⁶⁴ Intake is 0.82% of the dose.

  Importantly, the administration of immediate release zinc acetate capsules once a day does not provide adequate control of copper. As a result, the dose required for Wilson's disease patients is t.i.d., at a minimum b.i.d. Also, since zinc acetate capsules need to be administered at least 1 hour before meals and at least 1 hour after meals, the main problem is patient compliance. Administration once a day cannot be easily achieved by simply increasing the zinc dose, nor can it be achieved using sustained release formulations. The reason is that it is time dependent to induce enough metallothionein into the intestinal lumen to inhibit copper absorption. Exposure of the intestinal epithelium to zinc is typically estimated to take about two weeks to induce enough metallothionein to inhibit copper absorption when administered zinc more than once a day. The sustained release zinc formulation has several advantages as described below, but protects the transitional region of the intestine and is thereby absorbed over 6 hours.

  A further limitation on FDA-approved immediate release zinc acetate preparations is that the stomach becomes hypersensitive in connection with the release of bolus zinc cations.

<Gas retention formulation useful for maintaining copper Malabsorption>
Pharmaceutical dosage forms that remain in the stomach for a long time after oral administration and that release the active ingredient in a controlled manner are important for the delivery of a wide range of drugs. Drug delivery systems that control and release various drugs in the gastric residence time have been described in the literature. These documents are techniques different from the present invention.

  There are many advantages to using a drug delivery system. The main importance in controlled drug therapy is to increase the efficiency of the therapy. By controlling the drug treatment, the required number of doses is reduced and single doses at regular intervals are sufficient, thus improving compliance.

  In the case of current formulations of zinc acetate, it is necessary to administer 2-3 times or more per day in order to maintain poor copper absorption. This is because the action mechanism of zinc acetate induces the production of metallothionein in the gastrointestinal epithelial tissue, thereby inhibiting the subsequent absorption of copper from the region where metallothionein is expressed. Once expressed in gastrointestinal epithelial tissue, it is estimated that metallothionein binds and inhibits copper absorption for about 6-8 hours. Since the main part of copper, especially water-containing copper ions, is absorbed in the small intestine, it is necessary to refresh the small intestinal epithelial tissue several times daily so that the desired state of the small intestinal epithelial tissue can be maintained. .

  Therefore, an object of the present invention is to provide a preparation of zinc acetate using a gastric retention mechanism that can reduce the administration frequency of a drug once a day. The exposure time of zinc is continuously increased.

  The present invention can improve the level of copper malabsorption with the systemic exposure of zinc being equal to or lower than that of the prior art. It is. In addition to copper, zinc has been reported to bind to amyloid beta plaques in brains dissected from Alzheimer's disease patients. Some studies suggest that reducing zinc in the serum and CNS is a beneficial goal for the treatment of Alzheimer's disease [Bush AI]. However, the long-term effect of zinc in serum and CNS has not been evaluated in Alzheimer's disease. Therefore, the present invention reduces the uncertainties in the relationship between Alzheimer's disease and zinc by providing a means to inhibit copper absorption in the intestine while maintaining a minimum serum level of zinc. Given that the antioxidants and zinc present in Alzheimer's plaques may not have a causal relationship with copper, zinc may have a beneficial role in the serum and CNS.

  To improve the treatment of these diseases, gastroretentive pharmaceutical dosage forms utilizing a number of gastroretentive systems can be developed. In such gastroretentive dosage forms, after oral administration, it is maintained in the stomach and the release of zinc acetate is controlled over a long period of time. Gastric retention dosage forms include, for example, floating dosage forms and dosage forms that expand, expand, or open in the stomach.

  The principle of the inflatable drug delivery system is based on the nature of the pyloric sinus, and by reverse peristalsis, most is sent from the pylorus to the fundus and stomach, increasing the residence time (GRT) in the stomach. Such dosage forms are preferably biodegraded or undergo degradation that allows for elimination from the stomach.

  US Pat. No. 3,574,820 discloses the use of a gelatin matrix, which hydrates and gels in the stomach, crosslinks with N-acetyl-homocysteine thiolactone, and cannot pass through the pylorus Generate a matrix of size.

  U.S. Pat. The polymer envelope contains an inflating agent that, when in contact with bodily fluids, expands the envelope until the device is at a volume that is maintained in the stomach.

  The matrix comprising the device disclosed in U.S. Pat.No. 4,434,153 is formed from a hydrogel that absorbs, expands and swells fluid from the stomach so that it can be retained in the stomach for a long time, A small pill having a drug-containing core, a fatty acid, and a wax wall surrounding the core is dispersed.

  However, the problem with the devices in these patents is that they ignore the natural contractions of the stomach, which contributes to a rapid decrease in size, and the device is removed early from the stomach. In addition, these devices lack the strength to withstand natural mechanisms including gastric contraction.

  U.S. Pat. Flat devices with round protrusions), rings, etc. The device is compressible to a size suitable for swallowing and is self-expandable to details that prevent passage to the pylorus. They are sufficiently resistant to gastric forces and prevent rapid passage through the pylorus within a predetermined time and erosion by gastric juice. The device is homogeneous and contains the same polymer component in different regions of the device. The tetrahedron described in US Pat. No. 4,735,804 is homogeneous in four lobes and is attached to each other by a polymer matrix.

  The device contains zinc acetate as a solution or suspension. A solution or suspension is required to add the preservative or buffer. Alternatively, a controlled release zinc acetate module can be adhered to the device.

  US Pat. Nos. 5,002,772 and 5,443,843 disclose oral drug delivery systems that delay gastrointestinal transit, where the drug is controlled release and prevents gastrointestinal transit in an inflated state. These delivery systems include one or more retention arms as a non-continuous compressible element, and the controlled release drug-containing device is attached to the retention arms. The desired shape is a coil or a helix. These systems must have at least two separate parts (at least one holding arm and a controlled release device).

  U.S. Pat.Nos. 5,047,464 and 5,217,712 disclose systems that include bioerodible and thermoset, covalently crosslinked poly (ortho) ester polymers that are compressed during delivery. It expands from the state that it was. In the acidic environment of the stomach, the polymer in the system can degrade and be removed from the stomach. The system is characterized by high elasticity.

  US Pat. No. 5,651,985 is a system made from a mixture of polyvinyl-lactam and polyacrylate and has a high degree of swelling in the stomach and can stay in the stomach for a long time.

  U.S. Pat.No. 6,685,962 discloses a gastroretentive drug delivery system that controls the release of an active ingredient in the gastrointestinal tract, and (a) contains a polymer that does not retain more in the stomach than conventional dosage forms. A monolayer or multi-layer matrix comprising: (1) a degradable polymer that is hydrophilic but not readily soluble in gastric juice, substantially insoluble and enteric with a pH of less than 5.5 A polymer and / or a mixture thereof, (2) a non-degradable polymer and any mixture of (1) and (2), (b) a continuous having sufficient mechanical strength and comprising at least one polymer A membrane or discontinuous membrane, and (c) a drug that prevents the delivery system from being expelled from the stomach within a predetermined time by a matrix attached to the membrane.

  The pharmaceutical composition is prepared so that it can be administered by other routes (enema, nasal administration, subarachnoid administration, etc.) in addition to oral, intravenous, subcutaneous or inhalation administration. The advantages of oral dosage forms (solutions, suspensions, tablets, capsules, etc.) include fast therapeutic effects and patient convenience.

  In this field, oral administration has a direct effect on the target site in the gastrointestinal tract, and the therapeutic effect by absorption of the active ingredient when a pharmaceutical composition (acid neutralizer, laxative, etc.) is introduced into the patient's circulatory system. Is known to be different. Controlling the retention of solid drugs in the stomach is based on mucoadhesion, flotation, sedimentation, expansion mechanisms, or coadministration of pharmacological agents that delay gastric emptying. Can be done by.

  Mucoadhesion is the process by which synthetic and natural macromolecules adhere to mucosal surfaces in the body. When these substances are incorporated into the drug, drug absorption by mucosal cells is improved and the drug can be released over time at the target site. In the case of synthetic polymers such as chitosan, carbopol and carbomer, bioadhesion / mucoadhesion is the result of many different physicochemical interactions. Biological biomucoadhesion / mucoadhesion (eg, plant lectins) show interactions with cell surfaces and mucins and are recognized as “second generation” bioadhesive [Woodley, J., Bioadhesion: new possibilities for drug administration ?, Clin Pharmacokinet 2001; 40 (2): 77-84)]. Thus, mucoadhesion acts to impart the ability to resist the strong propulsive force of the stomach wall for oral dosage forms. The continuous production of the mucosa by the gastric mucosa replaces the mucosa damaged by peristaltic contraction, so that the dilution of the stomach components can be eliminated by using mucoadhesion as a means for staying in the stomach.

  Mucoadhesive nanoparticles (including liposomes and polymer nanoparticles) have been evaluated. The mucoadhesive ability imparted to the particle system is imparted by coating their surfaces with mucoadhesive polymers such as chitosan and carbopol. The feasibility of such surface modification has been confirmed by measuring zeta potential. The evaluation procedure includes particle counting using a polymer-coated liposome coulter counter. Mucoadhesive nanoparticles have been reported to be used for oral administration of peptide drugs and last longer in effect compared to systems without polymer coat [Takeuchi H., et al.]. Mucoadhesive nanoparticle systems are used for drug delivery of peptides [Adv Drug Deliv Rev, Mar. 23, 2001; 47 (l): 39-54].

  Mucoadhesive drug delivery systems have several advantages over conventional dosage forms, and the therapeutic effect of the drug can be optimized by controlling the release of the drug into the body. Various types of poly (acrylic acid) (PAA) hydrogels inhibit the hydrolysis of gastrointestinal enzymes (eg, trypsin) and improve the bioavailability of drugs. Acrylic-based polymers can be used to attach the mucoadhesive delivery system to the mucosa. A polymer hydrogel modified by grafting a parent mucosal copolymer (such as poly (ethylene glycol) (PEG)) onto the backbone of the polymer can facilitate the attachment process. This is because these graft chains can diffuse from the network to the mucosal layer. P (AA-g-EG) films are synthesized by polymerization of UV-irradiated free radical solutions. Different types of hydrogels can be synthesized with varying molar feed ratios of AA and PEG. Polymer hydrogels are characterized by mucoadhesion and the effect of PEG graft chains on mucoadhesion can be quantified. The bond strength of bioadhesion can be measured using a tensile test apparatus, whereby the adhesion capacity can be calculated. Hydrogels containing 40% AA and 60% PEG (40:60 AA / EG) can exhibit the highest mucoadhesive properties. These results are due to the synergistic effect of both monomers. The AA functional group allows the polymer to have multiple hydrogen bonds and increases the degree of expansion. The binding of PEG acts as a promoter that promotes mucosal adhesion. They penetrate the mucosa and bridge the base hydrogel and mucosa. These results are also explained in the Huang-Peppas model (2002) of surface coating and chain length effects in mucoadhesion.

  Flotation as a retention mechanism requires the presence of a liquid in which the administered product can float, and the patient must remain upright during GRI. This is because in the supine posture, the pyloric position is above the stomach body, which facilitates the discharge of suspended solids. Thus, flotation is the basic principle for retaining oral preparations in the stomach.

Deposition is used as a retention mechanism for pellets that are small enough to be retained in the gastric folds or folds near the pylorus. Note that the pylorus is a part of the organ located in the lowest position in an upright posture. High density pellets (approximately 2.4-2.8 g / cm ³ ) trapped in the folds or folds can withstand gastric wall peristalsis. The density of elemental zinc is 7.165 g / cm ³ , which is 4.365 g / cm ³ greater than the threshold 2.8 g / cm ³ required for gastric retention. The high density 7.165 g / cm ³ of zinc can create the opportunity to utilize up to about 60.9% of each pellet, incorporate other substances where low density is preferred, the function, safety, The durability and effectiveness can be improved. Therefore, the present invention discloses zinc pellets that are enteric coated on zinc pellets that are sufficiently small in size to be trapped in the jars or folds. Such enteric coating has the advantage of avoiding the risk of irritation of the stomach wall due to direct contact with the trapped zinc pellets, as described below. The enteric coating is pH dependent and is selected to prevent coating degradation and zinc release, and the pellet is in the low pH state of the stomach and / or pylorus (pH 1.2-3.5). On the other hand, in the duodenum (pH about 4.6-6.0), jejunum (pH about 6.3-7.3) and / or colon or rectum (pH about 7.9-8.0) with high pH, the coating begins to decompose and release zinc. Also, in the colon or rectum, it is based on the presence of the bacterial flora ubiquitous (at this site water containing high concentrations of copper is absorbed by the body). The pellets can also be encapsulated and rapidly dissolved and released when they enter the stomach. Capsules are optimized according to patient behavior and medication requirements, depending on the total amount of zinc included, the number and type of pellets included, and target “free” or “serum” copper is within a specified range. Can be changed to be maintained.

  Inflation is known to be a potentially reliable retention mechanism. Several devices have been disclosed in the art, which are characterized by being expanded, unfolded and expanded by carbon dioxide generated in the device after administration. These dosage forms cannot pass through the pyloric sphincter if the diameter in the expanded state exceeds about 12-18 mm.

  Therefore, it is an object of the present invention to provide a drug delivery system with controlled release so that the zinc acetate composition or device can be retained in the stomach for a sufficient time to release zinc acetate from the stomach. .

<Continuous protection from peak concentrations of copper-containing drinking water and "free copper">
Table 3 shows copper components present in common food products and various organs [Linder, MC [Handbook of Copper Pharmacology and Toxicology (2003), p. 4].

  As shown in Table 3, the limit proposed by the World Health Organization for copper in drinking water is 2,000 to 20,000 times the amount present in uncontaminated fresh water and is based on the Lead and Copper Regulation (2000 revision) It is 1,300 to 13,000 times the 1.3mg / L set in USEPA. At the limits of EPA, assuming an average daily drinking water consumption of 2 liters, drinking water will yield 2.6 mg of copper daily. Assuming an average daily intake of 1 kg, the amount of copper consumed in a daily meal is about 1.0 mg per day. However, in addition to copper bound to proteins contained in foods, a considerable amount of recycled copper entering the gastrointestinal system in already processed protein binding forms (saliva, gastric juice, intestinal secretion, epithelial cell scab formation and bile) It appears that the free, unbound, dissolved, potentially ionic copper contained in drinking water is processed by the intestine in a more enzyme-specific manner than copper. Since digestion to release the food-ligand complex is not necessary, a greater portion of the copper contained in the drinking water is absorbed as a bolus. Intestinal cells pass through a bolus of blood that is predominantly bound by albumin. Thus, due to the bolus effect, the main part of copper may be able to bypass the liver. That is, it can be captured by the brain, bypassing the intestinal copper absorption and regulation device.

  Copper in the drinking water enters the serum, binds to the small intestine, crosses the blood barrier and enters the central nervous system, bypassing the normal blood brain barrier regulation of copper transport and homeostasis. Free or weakly bound copper in the CNS and blood is considered to be in equilibrium. In addition to overwhelming the CNS with excess free copper, copper absorbed through drinking water is probably absorbed like a bolus compared to copper absorbed through food. The peak concentration of free copper in the CNS is expected to be significantly higher than the daily average. The peak concentration of free copper in the CNS probably up-regulates copper binding protein / protection mechanisms such as APP, amyloid beta and tau. Since the copper molecule exists in the form of ions, that is, cupric, the harmful potential is further increased.

  It is an object of the present invention to provide peak serum and CNS release by administering one or more special formulations of continuous release copper absorption inhibitors to copper sensitive patients (Wilson's disease, Alzheimer's disease, elderly dementia). It is to provide continuous protection from the toxic effects of copper concentration, thereby reducing the effects on harmful free copper, especially the up-regulating effect of protective copper proteins in the CNS.

  In a preferred embodiment, the gastrointestinal pill or capsule utilizes a combination of different gastrointestinal mechanisms to ensure the widest and least fluctuating protection. For example, in addition to high-density zinc salt pellets, the capsules may contain floating zinc salt-containing microparticles, mucoadhesive microparticles, mucoadhesive high-density zinc-containing pellets, mucoadhesive high-density enteric-coated zinc-containing pellets, zinc In addition to an expandable gastric retention system containing salt, it can be prepared with immediate release and / or non-gastric retention zinc powder or enteric coated zinc particulates. Such preparations are useful for physicians to estimate recommended daily intakes to obtain a level of copper protection or copper malabsorption.

<Enteric pills, capsules, tablets or fine particles containing zinc>
Zinc acetate formulations available as FDA-approved dosage forms (eg Galzin et al.) Are used for patients who cannot comply with gastric irritability. This is believed to be related to the ionic nature of zinc. It is estimated that about 10% of patients show gastric sensitivity to current formulations.

  Copper contained in copper-containing fluids (e.g. water) or food does not begin to absorb in the human body until it reaches the small intestine, thus inducing the expression of metallothionein in the stomach to maintain the copper absorption inhibition state do not have to. In addition to causing gastric hypersensitivity, zinc immediate release preparations intended to induce a state of copper absorption inhibition, as well as systemic circulating levels of zinc and circulation to other organ systems (e.g. the brain) Raise the level unnecessarily. Excess zinc levels in the brain and central nervous system contribute to certain neurodegenerative diseases that also involve excess copper. Examples of such diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis (ALS).

  Another object of the present invention is to utilize a zinc preparation with an enteric coating formed, and pills, capsules, tablets (or microparticles in tablets) containing zinc are used to complete the esophagus and stomach. The release of the zinc component is not started until it has passed.

  A typical example of an enteric coating is a polymeric material. Preferred enteric coating materials are bioerodible, gradually water-degradable and / or gradually water-soluble polymers. The “coating weight” or relative amount of coating material per capsule generally defines the time interval between digestion and drug release. Any coating is applied such that the entire coating does not dissolve in gastrointestinal fluids less than about pH 5, and is thick enough to dissolve above pH 5. In the practice of this invention, any anionic polymer that exhibits a pH-dependent dissolution profile can be used as an enteric coating to deliver the active drug to the lower gastrointestinal tract. The selection of the specific enteric coating material depends on the properties, which are resistant to dissolution and degradation in the stomach; impermeable to gastric juices and drugs / carriers / enzymes in the stomach; The ability to dissolve or degrade rapidly at the intestinal site of the target; physical and chemical stability during storage; non-toxicity; ease of application as a coating (intimacy with the substrate); and economic utility.

  Suitable enteric coating materials include, but are not limited to, hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, methylcellulose, ethylcellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimetate, hydroxypropylmethylcellulose phthalate, Cellulose polymers such as hydroxypropylmethylcellulose succinate and sodium carboxymethylcellulose; preferably acrylic acid, methacrylic acid, methyl acrylate, methacrylic acid, methyl acrylate, ammonium methyl acrylate, ethyl acrylate, methyl methacrylate and / or methacryl Acrylic acid polymers and copolymers made from ethyl acrylate (copolymer sold under the trade name "Eudragit") Rimmer); polyvinylpyrrolidone, polyvinyl acetate, polyvinyl acetate phthalate, vinyl acetate crotonic acid copolymers and ethylene - vinyl polymers and copolymers such as vinyl acetate copolymer; and shellac (purified rack (lac)) can be exemplified. Combinations of different coating materials can also be used to coat a single capsule. Particularly preferred enteric coatings used in the present invention are acrylic acid polymers and copolymers sold by Rohm Pharma (Germany) under the trade name “Eudragit”. Eudragit has a series of copolymers of E, L, S, RL, RS and NE, available as solubles, aqueous dispersions or dry powders solubilized in organic solvents. Eudragit's RL, NE and RS series copolymers are insoluble in the gastrointestinal tract but are permeable and are primarily used to sustain release. Eudragit series E copolymers dissolve in the stomach. Eudragit series L L-30D and series S copolymers are most preferred in the present invention because they are insoluble in the stomach and dissolve in the intestine.

  Particularly preferred methacrylic copolymers are Eudragit L, especially L-3O and Eudragit 100-55. In Eudragit L-30D, the ratio of free carboxyl groups to ester groups is about 1: 1. Furthermore, the copolymer is insoluble in the gastrointestinal fluid of the upper gastrointestinal tract having a pH of 5.5 or lower, generally pH 1.5-5.5, and generally easily dissolved in the lower gastrointestinal tract of pH 5.5 or higher. It is soluble or partially soluble. Another particularly preferred methacrylic acid polymer is Eudragit S, which differs from Eudragit L-30D in that the ratio of free carboxyl groups to ester groups is about 1: 2. Eudragit S is insoluble at pH 5.5 or less, but unlike Eudragit L-30D, Eudragit S is poorly soluble in gastrointestinal fluid at pH 5.5-7.0 (eg, small intestine). The copolymer is soluble at pH above 7.0 (generally the colon is). Eudragit S alone can be used as a coating for drug delivery to the large intestine. Alternatively, Eudragit S is not sufficiently soluble in intestinal fluid below pH 7, but it is prepared as a delayed release composition by using with Eudragit L-30D, which is soluble below pH 7, and the active agent can be used in various parts of the intestinal tract. Can be delivered to. Use more Eudragit L-30D or more Eudragit S to initiate delivery with more delayed release. One skilled in the art will appreciate that Eudragit L-30D and Eudragit S can be replaced with other pharmaceutical compositions having similar pH solubility properties.

  The enteric coating serves to control the release of the active ingredient, so that the drug release occurs at a nearly predictable location in the lower intestinal tract below where the drug release occurs without the enteric coating. Can do. Enteric coatings also prevent hydrophilic therapeutic agents and carriers from being exposed to oral, pharyngeal, esophageal and gastric epithelial and mucosal tissues and enzymes associated with these tissues. Thus, the enteric coating protects the active ingredient and the patient's internal tissue from any adverse effects before being released at the desired site to be delivered. Furthermore, the coated capsules of the present invention can provide optimized drug absorption, active ingredient protection and safety. Multiple enteric coatings made to release the active ingredient at various sites in the lower gastrointestinal tract make it possible to improve the duration of the delivery effect throughout the lower gastrointestinal tract.

  Usually, the coating contains a plasticizer for preventing the occurrence of pores and cracks that allow gastric juice to enter. Suitable plasticizers include, but are not limited to, triethyl citrate (Citroflex 2), triacetin (glyceryl triacetate), acetyl triethyl citrate (Citroflec A2), Carbowax 400 (polyethylene glycol 400), diethyl phthalate, citric acid Mention may be made of tributyl acid, acetylated monoglyceride, glycerol, fatty acid esters, propylene glycol and dibutyl phthalate. In particular, coatings composed of anionic carboxylic acrylic polymers usually contain about 10-25% by weight of plasticizers, especially dibutyl phthalate, propylene glycol, triethyl citrate and triacetin. The coating was also coated with other coating additives (e.g. detackifiers, antifoams, lubricants (magnesium stearate), etc.) and coating materials solubilized or dispersed. Stabilizers that improve the material (eg, hydroxypropylcellulose, acids and bases) can be included.

  In order to apply the coating to the capsule, a known coating method and apparatus can be used. For example, the enteric coating is formed into a capsule using a coating pan, an airless spray method, a fluidized bed coating apparatus, or the like. Detailed information on materials, equipment and processes for preparing coated dosage forms can be found in [Pharmaceutical Dosage Forms: Tablets, eds. Lieberman et al. New York: Marcel Dekker, Inc., 1989 And [Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 6.sup.th Ed. (Media, PA: Williams & Wilkins, 1995)]. As mentioned above, the coating thickness must be sufficient so that the orally administered formulation remains intact until it reaches the desired site for local delivery to the lower gastrointestinal tract.

<Copper maintenance treatment for elderly patients>
There are several reports of significant increases in serum copper, especially in elderly patients over 75 years old [Madaric A, Ginter E, Kadrabova J, Physiol Res. 43; 92: 107-11, 1994]. There are also reports on elderly patients with Alzheimer's disease [Squitti et. Al., 2005]. The general conclusion is that mammalian copper homeostasis is primarily controlled by excretion levels [Lindner MC, Biochemistry and molecular biology of copper in mammals, Handbook of Copper Pharmacology and Toxicology, edited by Massaro EJ. , Totawa NJ: Humana, 2002, pp. 3-32.]. Accordingly, it is an object of the present invention to achieve and maintain a healthy level of copper concentration in elderly patients by restoring normalization of liver copper excretion. In the past, while pursuing normalization of copper by blocking copper intake or chelating or complexing copper, the present invention normalizes copper excretion in the liver. Thus, by simply monitoring each patient's copper status, the copper status can be maintained within a target range, thereby avoiding copper poisoning and hypercopperemia.

  Examples of formulations useful for improving copper excretion in elderly patients include urciodiol, anti-inflammatory drugs, and anti-fibrotic drugs.

<Cumulative injection preparation of copper chelating agent, copper complexing agent and copper inhibitor>
An alternative to oral delivery formulations that reduce copper in the body is by means of subcutaneous injection or subcutaneous implant. Liposomes are known as general-purpose carriers for drug delivery. These carriers are generally made from lipids that are commonly present in multilayer systems and are biodegradable by normal metabolic pathways. Accordingly, these carriers are generally biocompatible. Multivesicular liposomes can be prepared with zinc and released into the body for over a month. Because these multivesicular liposomes are large in size (average diameter about 10-30 μm), they are not rapidly removed by macrophages, function as drug depots, and for a month or longer The drug can be released slowly over time. Such storage preparations improve adherence to Alzheimer's patients. Nevertheless, it easily induces sufficient amounts of metallothionein into the intestine to inhibit the absorption of copper from the intestine.

  Alternatively, subcutaneous implants have been developed and utilized so that drugs can be delivered over a period of one year or longer. Such implants generally utilize a polymer matrix that contains the desired drug and can be released in a time dependent manner. Osmotic pumps are used in the same way. Zinc-prepared implants can easily achieve adherence to medications, especially in Alzheimer's disease patients, and sufficient amounts of metallothionein are easily induced in the intestine to inhibit copper absorption from the intestine. The

  Also, transdermal patches can achieve the same purpose as implants and maintain zinc in the bloodstream in a steady state.

  In the case of sustained-release preparations, storage preparations, implants and transdermal patches, the addition of copper-inhibiting drugs such as zinc reduces the peak of weakly bound copper in serum and other body compartments. Benefits are provided. Therefore, the induction of copper transport proteins (eg, amyloid precursor protein, amyloid beta and tau) can be reduced by neurons at the time when intracellular copper levels are high. Thus, regardless of administration convenience, the system has the additional therapeutic effect of reducing the production of pathogenic copper transporters compared to the non-sustained mode, with an average dose of zinc Other copper inhibitors, copper chelators or copper complexing agents are delivered.

<Ascorbic acid-containing preparation>
High levels of ascorbic acid (vitamin C) have been reported to prevent copper absorption. The object of the present invention is therefore to utilize preparations containing ascorbic acid to inhibit the absorption of copper by the gastrointestinal tract.

<Diseases related to hyperhomocysteinemia>
Increased levels of serum homocysteine, a 135-dalton copper-binding protein, have been reported to be associated with Alzheimer's disease, atherosclerosis and schizophrenia [the Framingham Study, NEJM 2003]. Folic acid reduces circulating levels of homocysteine, but increasing its intake has been reported to help prevent the development of Alzheimer's disease [the Baltimore Longitudinal Group, 2005]. However, there is a daily intake limit beyond which the additional effects of folic acid cannot be expected. Homocysteine has the effect of maintaining harmful weakly bound proteins that can freely cross the blood barrier and may be an opportunistic pool of circulating low molecular weight copper-binding proteins.

  Therefore, it is an object of the present invention to provide schizophrenia caused by elevated levels of homocysteine upon administration of a copper chelator, a copper complexing agent or a copper digestion and absorption inhibitor (a gastric retention regulator of zinc, zinc and zinc salts). Is to reduce the circulating levels of copper-bound homocysteine in the serum and the CNS.

  The object of the present invention is to use zinc-cysteine complexes (such as U.S. Pat.No. 6,586,611 by Newsome) to treat neurodegenerative diseases (Alzheimer's disease, ALS) associated with Wilson's disease, schizophrenia, atherosclerosis and elevated free copper levels. And as a copper digestion and absorption disorder for the treatment of (including Parkinson's disease).

<Pharmaceutical formulations, preparations and methods that protect against harmful free copper fluctuations and peak levels related harm and maintain a stable free copper state>
There are many researchers and research groups, with the same opinion that copper and other metals (iron, zinc, manganese, aluminum) are associated with various neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, ALS, etc.) However, there is disagreement about the conclusion of whether copper and other metals in such diseases are excessive or insufficient. Therefore, suggestions on how to intervene them vary from research group to research group.

  For example, Alzheimer's disease lacks elemental copper, and treatment of Alzheimer's disease patients suggests a clinical process to replace copper [Bayer, Phinney and Westaway]. The idea is that the intracellular copper-binding proteins (such as APP and Aβ) should be up-regulated because the available copper by the nerve is withheld as a result of intracellular copper deficiency. In addition, because there is an excess of both copper and zinc in the brain of Alzheimer's disease patients, we recommend and test the use of clioquinol, which has the effect of binding copper and zinc in elderly plaque through the blood-brain barrier. There are reports [Bush, AI et. Al]. Recently, it has been reported that the effects observed in animal models are due to Cryokinol maintaining copper availability in the CNS [Bush].

  In addition, it has been reported that copper in drinking water promotes the formation of Alzheimer plaques in a rabbit model fed a high cholesterol diet [Sparks]. More recently, in Alzheimer's disease patients treated with statins such as the trade name Lipitor, there is a report that the clinical effect is due to the reduction of copper by statins, which is indicated by a decrease in ceruloplasmin levels [ Sparks, L, Dec. 2005]. In this report, the relationship between copper in drinking water and Alzheimer's disease has not been fully elucidated, but it is considered effective for patients with Alzheimer's disease to avoid copper-containing drinking water. In addition, serum levels of Alzheimer's disease patients and mild cognitive impairment (MCI) patients have increased levels of total copper [Squitti 2003] and increased levels of copper not related to ceruloplasmin [Squitti 2005] compared to the same age reference value. ) Has been reported. This is apparently inconsistent with reports that in previous studies [Snaedal] and other studies, levels of total copper and ceruloplasmin in patients with Alzheimer's disease were considered to be no different from the same age reference value.

  Another object of the present invention is based on observations that have not been reported so far, and the level of copper weakly bound to proteins other than ceruloplasmin (so-called free copper) in serum and cerebrospinal fluid (CSF) is: Based on substantial intraday fluctuations. While not being bound by any particular theory, the diurnal variation of free copper in the CNS is effective in elucidating contradictions regarding whether Alzheimer's treatment goals should be reduced copper supplementation or copper supplementation. It is.

  When the level of copper in the CNS, and thus the level of intracellular free copper, increases, proteins that can bind and deposit intracellular free copper (e.g., APP and cleaved transmembrane peptide Aβ) are up-regulated, Aβ-bound copper is deposited in the extracellular CSF. However, once free copper levels in the systemic circulation are reduced from peak levels (as a result of proper treatment and excretion by the liver into the bile and stool), APP upregulation ensures proper metabolic function of neurons that require copper. It is thought that the necessary available copper is temporarily deficient in neurons. In such cases, the copper binding protein (eg, tau) in the neuron is up-regulated to maintain an adequate supply of free copper available in the neuron.

  Furthermore, if the potential free copper in the serum and CNS fluctuates during the day, it gets worse immediately when ingesting water containing dissolved copper, copper ions and copper salts. Without being bound by any particular theory, such fluctuations in free copper are due to rapidly flowing copper-containing water, which passes through the blood-brain barrier via the small intestine, portal vein, serum, and plasma. , Sent to spinal fluid and brain. Unlike copper present in food, most of it is released gradually and becomes bioavailable over time as part of the normal digestive process of the intestine, but usually in tap water The dissolved copper is present in a free, unbound form, which enters the systemic circulation and CSF as a bolus immediately after entering the intestine with the first passage effect. Such copper influx has been reported to increase in the presence of sodium [Wapnir]. For this reason, the reaction on time by the up-regulation of intestinal metallothionein and liver ceruloplasmin cannot be performed, and the intestinal epithelium and liver copper regulation mechanism is bypassed or cannot be regulated. As copper-dissolved water enters, it deteriorates in the presence of sodium, which potentially links the intestinal sodium channel as a potential entry point for dissolved copper in the portal vein and liver circulation. Copper bonded to the kilomicron also enters the lymphatic circulation and eventually drains into the thoracic duct.

  Without being bound by any particular theory, hepatic dysfunction, known to decrease with age, is observed in elderly people who are susceptible to late-onset Alzheimer's disease and Parkinson's disease. As a result, the peak free copper concentration in the general circulation and CNS of the elderly does not increase with, for example, a bolus of dissolved copper contained in drinking water, but if such peak free copper concentration persists, impaired liver clearance Will be high for. Also, the area below the free copper curve (AUC) in the systemic circulation and CSF will be substantially greater than in adolescents who received a bolus of copper dissolved in drinking water.

<Experiment 1>
Four cohorts were prepared, each consisting of 12 or more patients. 1 cohort of late-onset Alzheimer's disease patients, 1 cohort of Parkinson's disease patients, 1 cohort of Alzheimer's disease patients and normals of the same age as Parkinson's disease patients, 1 containing the isotope Cu ⁶⁴ This is a cohort of normal people aged 20 to 40 years who were administered equal amounts of distilled water or tap water. Serum samples were obtained at time zero and every 20 minutes for 2-24 hours. Serum samples were fractionated into three or more bead-containing columns with binding specificity for ceruloplasmin, albumin and one or more other copper binding proteins (eg, homocysteine, apoE, etc.). . The beads were then separated separately with distilled water and the solution eluted for each was measured and quantified for total radioactivity by standard protocols. The protocol measures total radioactivity indicating the total solution volume and the total amount of Cu ⁶⁴ present in the solution on an absolute and percentage basis for both ceruloplasmin, albumin or other proteins. Alzheimer's and Parkinson's disease patients both had higher peak Cu ⁶⁴ values and AUC of Cu ⁶⁴ in albumin and other protein eluates compared to cohorts of the same age and 20-40 years old. In addition, the same age person showed both the peak Cu ⁶⁴ value and the AUC of Cu ⁶⁴ higher than those of 20 to 40 years old. The results obtained show that Alzheimer's disease, Parkinson's disease and the elderly have peak toxic effects as a result of dissolved copper in drinking water and as a result of prolonged exposure to free copper administered from drinking water. For the first time. If a continuous CSF sample is obtained, the results will be the same, and there will be free copper equilibrium between the systemic circulation and the CSF, so Wilson's disease patients with neurological symptoms will have systemic circulation. As well as an increase in the level of free copper, it is directly related to the clinical behavior and neuropathy observed in the patient.

<Experiment 2>
The same experiment as experiment 1 was repeated. However, the induction dose regimens for the four cohorts of Alzheimer's disease patients, Parkinson's disease patients, normal and young normals are as follows: (a) Rapid-release oral zinc acetate (trade name Galzin ) 100 mg / day was administered up to 14 days before Day 0. ; (B) 100 mg / day of gastric retention type oral zinc acetate was administered up to 14 days before 0 day. ; (C) 100 mg / day of gastric retentive oral zinc acetate and sustained release oral copper and / or iron (salt or plant fiber, howay, metallothionein, transferrin, dry milk, infant formula or natural copper or iron; 2 mg / day (conjugated to the binding additive) was administered up to 14 days prior to day 0. (D) oral tetrathiomolybdate (as an immediate release dosage form or preferably ammonium or other salt in a sustained release preparation) 2 μg to 120 mg / day at the same time or at midnight It was administered up to 1 hour before 14 days. ; (E) Some or all of the essential trace metals (zinc, copper, iron, calcium, molybdenum and manganese) were administered in excess of the daily recommended intake (RDA).

<Result>
Administration of the preparations described in Experiment 2 (a) to (e) had the effect of reducing the free copper peak and AUC in serum samples (and CSF) for all groups of patients.

Report on the use of copper supplements for the treatment of Alzheimer's disease patients based on the assumption that the level of available copper in the CSF and / or CSF of Alzheimer's disease patients is lacking for normal neurometabolic function [Kessler H, Pajonk FG, Supprian T, Falkai P, Muthaup G, Bayer TA]. There is a report on the role of copper in the pathophysiology of Alzheimer's disease [Nervenarzt, 2005 May; 76 (5) 581-5 and Bayer TA, Multhaup G]. There are reports on the regulation of copper homostasis by amyloid beta precursor protein (AbetaPP) [J. Alzheimers Dis. 2005 Nov; 8 (2); 201-6; discussion 209-215]. The results of the above experiment can be reproduced by replacing the Cu ⁶⁴ supplement described in Bayer with the aforementioned Cu ⁶⁴ containing drinking water. The immediate release preparation described by Bayer also increases free copper peaks and AUC levels in systemic circulation and CSF patients. Since it is the same in Alzheimer's disease that neuronal copper deficiency occurs within a day, the prior art cannot identify free copper peaks and AUC daily fluctuations. In addition, the first-pass effect of bolus administration of liver free copper leads to peak levels of free copper uptake into the liver and excretion from the bile duct, resulting in neuronal copper deficiency. Temporary overproduction cannot be specified by the prior art. The combined copper, zinc iron and other trace metal preparations are released gradually, with the advantage that copper, iron and trace metals can be properly ingested, thereby substantially reducing neuronal load and compensation Metallic proteins (eg, APP, Aβ, tau, BACE1 and apoE) are produced.

<Experiment 3>
Experiment 3 relates to the replenishment of immediate release copper and sustained release copper.
The first cohort of 24 Alzheimer's disease patients was administered the immediate release copper supplement (containing 2 mg copper) described in Bayer TA once a day for 1-3 months. A sustained release preparation containing the same amount of copper was administered once a day for the same period to a second cohort of 24 Alzheimer's disease patients. The sustained release preparation is a salt, or preferably a salt bound to a natural copper binding carrier (eg, metallothionein, fiber, howay, casein). During the experimental period, all patients withdrew copper-containing drinking water and were allowed to balance their daily intake of copper-containing food and the use of cholesterol-lowering agents and other drugs. Administration of continuous serum samples was performed at weekly intervals of at least 12 hours after meals and within 1-3 hours after daily dose administration. In the same manner, by switching alternately between 12 hours and within 2 hours after administration, free copper (using the direct measurement method of free copper described above as the free copper measurement method), total copper, ceruloplasmin and 24S The level of -hydroxy-cholesterol (which is thought to be produced in the brain and increases in the serum of Alzheimer's patients) was examined.
<Result>
Cohorts treated with immediate release copper had higher free copper peaks and calculated estimated AUC levels in the serum of patients with Alzheimer's disease than cohorts administered sustained release copper, but serum levels of total copper and ceruloplasmin There was no statistically significant difference. Most importantly, however, for the time-weighted average of 24S-hydroxy-cholesterol, the cohort with immediate release copper was significantly higher than the cohort with sustained release copper, The progression of oxidative disease in the CNS. This effect is supported by the crossover design.

<Experiment 4>
The present invention relates to gastric retention type sustained release zinc.
A double-blind, placebo-controlled clinical trial was conducted in patients with Alzheimer's disease. Cohort I was placebo, Cohort II was administered 2 mg / day of immediate release copper supplement, Cohort III was administered 100 mg / day of gastric retention type sustained release zinc acetate (no copper supplement), Cohort IV was Stomach retention type sustained release zinc acetate was administered at 100 mg / day and sustained release copper supplement was administered at 2 mg / day, and the implementation period was 12 to 24 months. Patients are not restricted to drinking regular tap water or bottled water, but normal habits and common concomitant medications (e.g. statins (such as atorvastatin), cholinesterase inhibitors (such as donepezil)) and NMDA receptor antagonists (Memantine etc.)) was balanced.
The primary endpoint of the study is clinical improvement based on the mini-mental state test (MMSE) score, brain atrophy, volumetric measurement by MRI (minimum resolution 1.5T) and proton magnetic resonance spectroscopy (PRESS-J and automated 1H-MRS using repositioning technique [see Hancu I, et. Al (2005)]).
<Result>
Cohort III had the least decline but showed a higher incidence of hypocopperemia than Cohort IV. Cohort IV had less decline than Cohort I or II, and Cohort II had the greatest decline in primary and secondary endpoints.

<Low dose tetrathiomolybdate>
The object of the present invention is to provide a low dose pharmaceutical composition of tetrathiomolybdate that is effective in the treatment of various diseases. To the best of the inventors' knowledge, tetrathiomolybdate doses below about 0.25 mg / kg have never been tested and used in noble animal models or human diseases. Such low dose treatment is a significant departure from previously utilized concepts. In previous experimental and clinical settings, commonly used doses have been about 1 mg / kg / day to 1.5 mg / kg / day, so that the level of available free copper is negligible. The amount is reduced. This dose is titrated to maintain available free copper levels within a specific therapeutic range. It should be noted that copper bound to a ternary complex of tetrathiomolybdate and albumin is difficult to discriminate by means currently in practical use, so the level of free copper is at the whole body level of ceruloplasmin. Measured indirectly based on.

  Based on the observations regarding substantial diurnal fluctuations in free copper peak values (measured using the direct measurement technique described above), the purpose of the present invention is to reduce serum tetrathiomolybdate at low and stable concentrations. Free copper derived from free copper flux (from drinking tap water containing soluble copper) is combined with copper dissolved in tap water to form a ternary complex with serum albumin However, by making available to low molecular weight proteins such as homocysteine that can cross the blood-brain barrier, the level of free copper in the serum is lowered and stabilized. Copper bound to thiomolybdate and albumin is generally too heavy to pass the blood-brain barrier, and copper complexing agents (such as tetrathiomolybdate) contain copper chelating agents (d -Unique pharmacological effects as compared to penicillamine, trientine, criquinol, EDTA and other metal complexing agents. Free copper bound to low molecular weight proteins (such as homocysteine) readily crosses the blood-brain barrier and places a burden on the intra-neuronal and extraneuronal copper protein mechanisms necessary to maintain copper homeostasis at an appropriate level. This is because it is applied. In the case of Alzheimer's disease, low-dose sustained-release tetrathiomolybdate preparations up-regulate copper-binding regulatory proteins (such as APP, Aβ and tau) by stabilizing the level of free copper in the systemic circulation And has an important therapeutic effect of minimizing production. This unique mechanism of action and important advantages cannot be expected with copper chelators. The reason is that the copper chelator reversibly binds copper from various compartments. In addition, as described above, previous studies on copper-binding proteins that can cross the blood-brain barrier have misdirected the whole, and the mechanism of action in the present invention has not been suggested by conventional knowledge. . Drugs such as trientine and criquinol appear to be little or not effective in stabilizing CNS free copper levels. Also, preparations such as d-pensilamine and trientine non-selectively chelate copper from other organ compartments such as the liver that are not CNS, so for example, in neurogenic Wilson disease, CNS free copper It turns out that it raises a level. In Parkinson's disease, when a low dose of thiomolybdate, such as tetrathiomolybdate, has the effect of stabilizing the CNS free copper to a low level. Again, up to now, free copper levels have increased in Parkinson patients, and the production of copper and iron-binding protein α-synuclein (a major part of Lewy bodies is associated with Parkinson's disease and other synucleinopathies). It was reported to be down-regulated. This advantage is further enhanced when used with iron complexing agents and / or sustained release iron supplements that have the inducing action of protective metalloproteins (such as transferrin) in the intestine and liver, thereby allowing for soluble iron in tap water. It can protect against free iron bolus concentrations by free iron influx and immediate release iron supplement preparations. ALS also reduces the load applied to the copper scavenger Cu / Zn superoxide dismutase (SODl). In familial ALS, a genetic deficiency occurs in its copper binding protein. The same is true for copper-binding apoliprotein E, with variant 4 (apoE4) lacking two types of copper-binding cysteine and limiting the ability of apoE to exclude copper from the CNS by the liver.

  With an important consideration of the present invention regarding the therapeutic purpose of stabilizing systemic and CSF levels of free copper, experts in the field will be able to develop a variety of other new formulations. It will be possible. The preparations include, for example, copper binders bound to various proteins such as PEGylated peptides containing cysteine residues such as SOD1 and copper and iron binders of liposome complexes (tetrathiomolybdate, more specifically For example, sustained-release tetrathiomolybdate, and more specifically, low-dose sustained-release tetrathiomolybdate that can achieve the same function as reducing and stabilizing free copper) . Thus, the object of the present invention is to provide a method of using any formulation that can bind and render free copper available, said formulation being available in a stable state in serum, Preferably, the preparation does not cross the blood brain barrier. One aspect of the present invention is to provide a stable immediate release pharmaceutical composition containing thiomolybdate useful for chelating and complexing copper in the stomach, gastrointestinal tract and serum. The pharmaceutical composition and method described in this specification are useful for inhibiting copper absorption from the intestine and complexing serum copper to excrete from the human body. In particular, the pharmaceutical composition has the action of complexing and sequestering copper in food and beverages, and copper excreted from saliva, stomach, pancreas, bile ducts and from intestinal cells after eating, Thereby, while reducing copper absorption, when separated from food, free copper in the serum can be complexed to reduce the concentration of free copper. The present invention provides a rapid release that can be rapidly dispersed in the stomach to overcome the inherent instability associated with thiomolybdate so that the copper in food and beverages can be complexed and sequestered after meals. It is to provide a sex thiomolybdate preparation. The thiomolybdate preparations of the present invention have a commercially acceptable shelf-life, and after the meal, gastric and intestinal, without compromising the rapid dissolution properties necessary to adequately disperse thiomolybdate. Among them, copper can be complexed and sequestered. The present invention is a stable immediate release preparation and method that, when separated from food, can chelate and sequester copper in the patient's serum through the gastrointestinal digestive wall. The preparation of the present invention can reduce the dosing frequency while maintaining the copper reducing therapeutic action of the whole body.

<Thiomolybdate>
Thiomolybdates are composed of molybdenum and sulfur and include, but are not limited to, (MoS ₄ ) ₂ and (MoO ₂ S ₂ ) ₂ . Thiomolybdate can be made as a pharmaceutically acceptable salt (eg, a diammonium salt). These molecules can act as bidentate ligands to make copper complexes. Examples of thiomolybdate include, but are not limited to, tetrathiomolybdate, trithiomolybdate, dithiomolybdate and monothiomolybdate. Another example is complex thiomolybdates, including but not limited to zinc or iron between two thiomolybdate groups, which can bind and complex copper. Includes a date. In the example of a complex thiomolybdate, the molecule can have 5 or more thio groups with respect to 2 or more molybdenum. The Group VI transition metal tungstate can be substituted for the molybdate of the preparation as a thiotungstate.

A particularly relevant thiomolybdate for the treatment of Wilson's disease presenting with neurological symptoms is tetrathiomolybdate. Tetrathiomolybdate has molybdenum surrounded by four sulfurs (MoS ₄ ) ₂ .

  Since the 1980s, oral tetrathiomolybdate has been the subject of preclinical courses and human clinical trials [George Brewer, MD, Fred Askari, MD], as well as other treatments for neurologic Wilson disease with early symptoms. Others have been used in treatments that benefit from inhibition of intestinal copper absorption and the sequestration and removal of copper endogenous to the human body.

  Nervous Wilson disease is a genetic disease caused by mutations in the ATP7B gene and is characterized by a reduction in liver function that takes up copper into the ceruloplasmin protein and excretes copper via bile and stool. This reduced function increases liver and brain copper concentrations, and the increase in free copper in the systemic circulation of Wilson's disease patients is detrimental to the liver, brain and other organs.

  The tetrathiomolybdate regimen used for Wilson's disease patients with early symptoms is a treatment period of up to 16 weeks, and patients are sometimes given zinc (eg, zinc acetate) at the same time. The treatment also includes daily maintenance treatment, and a zinc-containing preparation such as zinc acetate (trade name Galzin) is administered. During a treatment period of up to 16 weeks with tetrathiomolybdate, tetrathiomolybdate is administered orally with food up to 3 times a day, with additional oral administration separate from food. The goal of tetrathiomolybdate taken with food is to act in the stomach and intestine, complexing copper in endogenous copper that enters food, beverages and the gastrointestinal tract, making it unavailable for systemic absorption That is. Tetrathiomolybdate is separated from food and promotes its systemic absorption, allowing it to form a ternary complex of serum proteins (such as albumin) and harmful free copper in the serum, thereby causing harmful effects in the serum. It reduces the availability and concentration of free copper and the availability to the central nervous system (CNS). Decreasing availability of copper to the CNS can reduce the level of free copper to normal levels, thus preventing or treating psychopathological and neurodegenerative diseases due to increased free copper in the CNS .

  Tetrathiomolybdate is unstable in temperature and humidity under typical atmospheric conditions, and the active ingredient is oxidized to molybdenum sulfoxide. Molybdenum sulfoxide is not active as a copper binder and therapeutic agent that lowers copper levels in the human body. Tetrathiomolybdate is believed to lose about 10% of its effectiveness within 3 months when stored under such conditions.

  The oral thiomolybdate drug can be rapidly released in the stomach and rapidly dispersed to complex copper in food and beverages, as well as acceptable stability and uniformity of efficacy over the lifetime Achieving the two goals of making things has been considered a previous subject in the field. For example, U.S. Patent Application No. 11 / 256,540 (filed October 21, 2005, Ternansky et.al) describes thiomolybdate that directly incorporates thiomolybdate into a matrix material (corresponding to diamonds) for improved stability. A solid dosage formulation of the compound is described. However, this has the serious drawback of slow release and dispersion of thiomolybdate in the stomach and cannot achieve the desired copper complexing effect with thiomolybdate in the stomach. US patent application Ser. No. 10 / 447,585 (filed May 28, 2003) discloses thiomolybdate analogs having pharmaceutically acceptable salts and esters other than ammonium. Such analogs have some improvement in stability compared to ammonium tetrathiomolybdate, which is most clinically used in Wilson's disease, but completely eliminates the stability problem in humid atmospheres. There is no advantage over ammonium tetrathiomolybdate.

  One aspect of the present invention relates to an immediate release capsule containing thiomolybdate, the capsule having a very low moisture content and containing a low moisture additive, the capsule packaged in a container or blister pack The capsule is purged and sealed with an inert gas (such as nitrogen or argon) to protect the thiomolybdate from atmospheric oxygen. Capsule packaging may also include one or more chemical indicators. The indicator is, for example, an indicator for moisture or oxygen, and a warning is issued by breaking the anhydrous or anaerobic seal.

  The present invention also provides a method of administering thiomolybdate immediately prior to ingesting a meal and an appropriate amount of beverage so that copper containing foods and beverages can be rapidly degraded to be most widely protected by the complex. To do.

  The present invention also includes the use of low doses of sustained release tetrathiomolybdate or other equivalent such formulations, in addition to the CNS, the free copper peak and oxidation by elevated AUC are known to play a role. To treat other diseases (eg, atherosclerosis and liver disease (NASH, etc.), non-viral hepatitis and diabetes).

  In order to maximize the bioavailability of a sustained release preparation of intact tetrathiomolybdate, the sustained release preparation may be composed of, for example, two or more particulate forms contained in a single pill capsule or tablet or Matrix types can be preferentially included. Such pills or capsules can include enteric-coated particles in sustained release dosage forms, which are designed to be released, for example, in the pH environment of the jejunum and small intestine, and also the ileum And is designed to be released in the colonic environment. Such preparations and methods are known in the art, but the mechanism of action of tetrathiomolybdate depends on the oxidation of the sulfur group to its copper binding and albumin complexing properties (hence the initial oxidation). Such a preparation has the advantage that it limits the exposure of the tetrathiomolybdate contained therein and prevents oxidation prior to release.

  In order to further limit oxidation and improve the stability of tetrathiomolybdate, the present invention can also include a pharmaceutical package, whereby each pill, tablet or capsule is provided for use. Until packaged and sealed separately in a controlled atmosphere. Packaging is, for example, an impervious foil pouch or a cold formed blister pack that is purged with argon or nitrogen gas to remove more than 98% of the atmosphere and moisture. If capsules are used, the capsules with tetrathiomolybdate should be selected with a particularly low water content so that the interaction between the capsule and the active ingredient can be limited. The pouch or blister pack packaging can be packaged with desiccant and sacrificial oxidant in a package purged with an inert gas to improve stability.

  In a particularly preferred embodiment of the invention, the crystal size of tetrathiomolybdate is controlled so that release in the stomach and intestine is selected, sustained and delayed. The crystal size is controlled by controlling crystal growth or by milling described later. The inventors found that large crystals of tetrathiomolybdate (e.g. ammonia tetrathiomolybdate) dissolve essentially in the stomach and dissolve in the intestine, while small crystals dissolve in the stomach. I found out. This makes it possible to achieve a desired sustained release property by using a mixture of crystals having different sizes. For example, a mixture of 50-100 microns and 200-500 microns can be included in a single capsule.

  The inventors have found that the kinetics of ammonium tetrathiomolybdate (ATTM) in artificial gastric juice depends on the crystal size of ATTM. ATTM crystals with a size of 50 microns or less (cross-sectional width) dissolved rapidly in the acidic buffer (pH 2). After 1 hour culture in acidic buffer, ATTM was gone. However, when the ATTM crystal size was greater than 1700 microns, dissolution in acidic buffer was slow. After incubation for 1 hour in acidic buffer, ATTM remained over 50%.

<Method>
1. ATTM was purchased from Sigma-Aldrich and sieved through a 1700 micron screen. Crystals that have not passed through the screen are referred to in this experiment as “large”.
2. The ATTM crystals were ground to reduce all crystals to 50 microns or less. These crystals are referred to as “small” crystals.
3. Each crystal was weighed 100 mg and added separately to a 1.7 ml plastic centrifuge tube.
4). 1 ml of pH 2 buffer was added to each tube without mixing.
5. After 1 hour, each tube was centrifuged for 1 minute at 14,000 rpms.
6). The supernatant was removed and the ATTM was analyzed.
7). To each tube pellet, 1 ml of pH 7.4 phosphate buffer was added.
8). Each solution was vortex mixed.
9. Each tube was centrifuged for 1 minute at 14,000 rpms.
10. The tops were taken from each tube and analyzed for ATTM.

<Analysis>
1. 100-fold dilutions for all supernatants were prepared in pH 7.4 phosphate buffer.
2. 2 ml of each diluted sample was added to a plastic cuvette.
3. Absorbance was measured at 467 nm.
4). The ATTM extinction coefficient (EC) was calculated for each sample.
of ATTM for each sample were calculated.

<Result>

<Observation>
1. When acidic buffer was added to the small crystal ATTM, the solution immediately turned black and a sulfur scent was detected.
2. The black solution eventually became a black precipitate and deposited at the bottom of the tube, and the supernatant became clear. This was observed after 1 hour of culture.
3. When acidic buffer was added to the ATTM large crystals, a small amount of black leached from the crystals. Furthermore, the formation of a black coating was observed on the outside of the crystal, which seems to have prevented further dissolution.

<Conclusion>
1. In the case of small crystals, ATTM dissolves in an acidic medium but eventually forms an insoluble precipitate.
2. Dissolved ATTM does not remain in the acidic medium.
3. In the case of large crystals, ATTM forms a black coating in the acid, which provides resistance to dissolution.
4). Undissolved ATTM is liberated from large crystals in acid and secondarily dissolved in neutral medium.
5. The black coating does not appear to have an anti-dissolving effect in neutral media.

<Experiment 5>
Large (> 1000 microns) and small (<50 microns) dark orange crystals of tetrathiomolybdate were placed in commercially available gastric juice simulation solutions. Large crystals released a slight sulfur scent. The crystals themselves turn relatively black without being compromised, but the solution remains transparent. When a small crystal is placed in the same kind of solution, a strong sulfur scent is immediately released and the composition is a black turbid solution. The difference in dissolution characteristics between large crystals and small crystals is obvious. Furthermore, the characteristics of methylcellulose capsules containing 20 mg of tetrathiomolybdate and 180 mg of additive containing tetrathiomolybdate crystals in different size ranges (200-800 microns), initially containing simulated gastric acid Tested in a dissolution chamber. Gelatin capsules dissolved within 15 minutes. The recovery of tetrathiomolybdate was evaluated periodically over 3 hours by UV-visible absorption. This time is time consistent with the onset of gastric emptying and the initial phase of postprandial absorption. No tetrathiomolybdate was detected in simulated samples of stomach removed and neutralized at any time during this 3 hour period. At 3 hours, the remaining gastric juice became alkaline by adjusting the pH to 8-9, and the solution changed from orange to red and was evaluated by UV-visible spectroscopy. Tetrathiomolybdate recovery was approximately 7% of the capsule volume.
The same experiment was performed on small crystals, but recovery was much less than 7% of the parent compound, whereas when the additive was performed on large crystals, it was much more than 7% of the parent compound. To be recovered. These experiments indicate that the crystal size of tetrathiomolybdate is a determinant to protect against gastric acid denaturation.

<Experiment 6>
Tetrathiomolybdate crystals are ground to a powder (in this case, initially orange) or dissolved in water to form a bright orange solution, which is then lyophilized through a filter. To a dry orange powder. The ground powder is purged with a nitrogen overlay and slowly turns black within a few days at room temperature in a closed tube, while a dry powder that has been made into a dry powder under the same conditions is more than a month It is an orange cocoon over the period. This experiment shows that when sufficient water is removed from tetrathiomolybdate, stability to the atmosphere is improved. Similarly, mineral-coated orange tetrathiomolybdate is protected from the atmosphere and moisture and is orange (ie, unoxidized) wrinkles for one month.

<Experiment 7>
An equal amount of powder of lyophilized tetrathiomolybdate is dissolved in water, encapsulated, prepared as an uncoated tablet or enteric-coated tablet, once a day for fed or fasted rats. Administered over a week. At the end of the multiple dosing period, blood samples were taken periodically over a 24 hour period to make ceruloplasmin pharmacodynamic and molybdenum drug conductor determinations. Both fasted and fed rats, which received enteric coated tablets, showed ceruloplasmin decreased to the maximum possible and serum molybdenum with the greatest increase.

<Experiment 8>
This experiment is for low dose tetrathiomolybdate.
Double-blind, placebo-controlled clinical trials in patients with Alzheimer's disease to compare placebo (Cohort I) with low-dose (2-10 mg / day) sustained-release ammonium tetrathiomolybdate (Cohort II) up to 24 Done for months. The primary endpoint of this experiment is clinical improvement based on assessment of cognitive abilities such as mini-mental state test (MMSE) scores, and brain atrophy is measured by MRI (minimum resolution 1.5T) volume measurement and proton magnetic resonance spectroscopy. (1H-MRS using PRESS-J and automatic repositioning technology [see Hancu I, et. Al (2005)]).
<Result>
Cohort I had the least decline of all parameters, and there was less incidence of hypocopperemia than Cohort II.

<Experiment 9>
A gastroretentive sustained-release maintenance treatment with tetrathiomolybdate was performed.
A double-blind, placebo-controlled clinical trial was conducted in patients with Alzheimer's disease to compare placebo (Cohort I) with an induction dose (80-120 mg / day) of immediate release ammonium tetrathiomolybdate. Immediate release ammonium tetrathiomolybdate is described in the inventor's U.S. Patent No. 6,855,340 and patent application No. 10 / 444,204, and after administration for 3-6 months, gastroretentive sustained release zinc is applied. Maintenance therapy with 50-100 mg daily was given for an additional 18-21 months. Whether or not a sustained release copper supplement is necessary was determined based on whether the systemic ceruloplasmin level was lower than the target level (for example, 12 mg / dl or less) and / or there was a sign of hypocopperemia. The primary endpoint of this experiment is clinical improvement based on assessment of cognitive abilities such as mini-mental state test (MMSE) scores, and brain atrophy is measured by MRI (minimum resolution 1.5T) volume measurement and proton magnetic resonance spectroscopy. (1H-MRS using PRESS-J and automatic repositioning technology [see Hancu I, et. Al (2005)]).
<Result>
Cohort II shows less reduction than Cohort I in all parameters, and improves brain metabolic function compared to Cohort I in a short period of 3-6 months.

<Molybdenum and sulfur-containing preparation>
It is an object of the present invention to provide an oral supplement preparation containing elemental molybdenum and sulfur. When this preparation is administered and dissolves in the stomach, it binds in the stomach of a low pH environment to produce thiomolybdate, which can complex free copper in the gastrointestinal tract and systemic circulation. Yes (mostly ammonium tetrathiomolybdate). Such preparations are much less reliable in terms of medication, but may have advantages as natural products based on the Dietary Supplement Education Bill (DSHEA).

  For use in therapy, the sustained release trace metals of the present invention are in the form of pure cations or salts, pharmaceutically acceptable sustained release microspheres, matrices, pellets or particles (all in the art). But first of all natural or synthetic carriers (e.g. plant fibers) with pharmaceutically acceptable stability known to be able to bind such metals. , Howay, metallothionein, transferrin, protein and / or milk or milk by-product). Since trace metals are naturally contained in food, such carriers have the effect of inducing slow digestion and absorption of trace metals. Thus, the present invention includes one or more trace metals (eg, zinc, copper and iron) along with one or more pharmaceutically acceptable carriers, and optionally other therapeutic and / or prophylactic agents. Further provided are gastroretentive and / or sustained release pharmaceutical preparations. The carrier is acceptable in terms of compatibility with the other ingredients of the preparation and is not detrimental to the user.

Another object of the present invention is to provide a copper supplement that can be administered with a copper malabsorption agent, whereby the copper is bound to a lipid, howay or casein dosage form. Or a dosage form prepared in lipid, howay or casein. Copper supplements prepared in this way are provided in the form of pills, capsules or liquids, which are similar to the supply of copper from mother's breast milk to infants. Therefore, unlike copper contained in the form of water or pure salt, such copper reaches the stomach and small intestine for proper digestion. The bioavailability of such copper supplements is as high as 24% of breast milk and 18% of milk, and can be normally digested and processed by the small intestine and liver, so serum and CNS The level of burden of free or weakly bound copper in it can be reduced. The preparation can also include other essential metals or minerals such as iron or zinc.
In a preferred embodiment of the present invention, such carrier-bound copper supplements can be included in sustained release microparticles or matrices to allow further regulation of absorption and free copper in the systemic circulation and CSF. Reduce the ability to produce peak elevated levels. Such preparations can reduce or avoid the need to monitor patients with hypocopperemia or anemia and reduce the level of serum or CNS free or weakly bound copper. This method of copper and metal supplementation has proven to be evolutionary, and the treatment of the liver with first-pass effects and normal copper treatment is due to the fact that the mother nourishes the newborn through breast milk and other It is similar to supplying and processing metal. Such preparations can also include other essential trace elements (eg, iron, calcium, molybdenum, selenium, magnesium, etc.) in a sustained release complexed preparation with a carrier bound thereto. Complexed sustained release calcium preparations are useful for the treatment or prevention of diseases involving calcification, for example, in arteries, kidneys, lungs and brain.

In a preferred embodiment of the present invention, the stable isotopes Cu ⁶⁵ and Cu ⁶⁷ are used as active copper components so that copper balance and therapeutic effects can be assessed and adjusted on an individual basis. This study can be done by collecting serum, CSF fluid, stool or urine, or by biopsy. Such information provides useful diagnostic methods, evaluates the effects of other treatment methods (such as therapy), and improves the excretion of copper by the liver and the action of copper chelators.

  The copper and zinc preparations described in this specification can be administered parenterally as a nutrient, by immediate release, or by cumulative injection. Oral nutritional preparations of zinc and complexed copper can include certain copper absorption enhancers such as glycerol and NaCl, or gum arabic to improve the bioavailability of complexed copper.

  The copper and zinc preparations described in this specification are also useful for gastrointestinal diseases such as chronic diarrhea, diarrhea-type irritable bowel syndrome and infections for which zinc is known to be useful.

  The present invention can be administered as a two-pill system, where the complexed copper pill or preparation is first administered orally, and then the zinc-containing pill is orally administered together or after a sufficient time.

  The present invention also contemplates the inclusion of other essential minerals where zinc in the intestinal tract can reduce bioavailability, such as iron and cadmium.

  The present invention can be preferentially combined with cholesterol lowering agents for the treatment and prevention of atherosclerosis, dementia, Alzheimer's disease and other neurodegenerative diseases. Neurodegenerative diseases include neuromuscular diseases associated with abnormal accumulation of copper-related proteins, such as juvenile and sporadic inclusion body myositis and elderly myositis.

  A further embodiment of the present invention is the use of a zinc preparation or copper lowering agent as described herein for the treatment of neurological and psychiatric signs of liver disease associated with inhibition of hepatic copper excretion, said liver Diseases include diseases in which free or weakly bound serum or CSF copper is elevated, such as cholangitis, hepatitis and cirrhosis.

<Experiment 10>
A pill or capsule containing 100 mg of high density zinc salt pellets that can stay in the stomach wall is administered to the patient group in the morning. The control group takes normal zinc acetate 100 mg, and another control group takes placebo. These patients take distilled water or tap water containing copper or Cu ⁶⁴ at the same time throughout the day. Serum samples are collected before, during or after ingestion of the beverage. This sample was immediately measured for each free copper or weakly bound copper. The measurement is known by a method of subtracting copper bound to ceruloplasmin from total serum copper, or by a separation method based on molecular weight according to a column method [Bohrer D (2004) or MALDI-TOF described by Sarkar E (2004)]. Alternatively, it was performed by a method of directly measuring copper bound to unknown proteins and peptides.

<Result>
The placebo group showed the highest peak level for free or weakly bound copper, the bolus oral zinc group showed the lowest peak level for free or weakly bound copper, and the gastroretentive sustained release zinc group was free The lowest level was shown for copper or weakly bonded copper. To reveal long-term benefits for intestinal metallothionein induction to inhibit copper absorption, experiments were repeated daily until statistical significance was achieved.

<Experiment 11>
Copper, copper zinc, or copper bound to low molecular weight amino acids is dissolved in a solution of dry milk or concentrated milk, dry howay, dry milk fat, milk protein or other natural copper binding protein. The resulting mixture of copper bound complexes can be dried and prepared as pills or tablets. In a preferred embodiment of the invention, the dried copper binding complex is prepared with zinc in a pill or capsule. In other preferred embodiments, pills or tablets are prepared using gastroretentive dosage forms of zinc, enteric coated zinc and / or sustained release zinc, the copper binding complex prior to zinc, Released into the gastrointestinal tract. Copper binding complexes are digested in the gastrointestinal tract, absorbed by enterocytes, and processed intracellularly by metallothionein and / or transcuprein. Here, the copper to which such metallothionein and / or transcuprien is bound enters the liver, is taken up by ceruloplasmin, released into the serum, stays in the liver, or is excreted into bile. The zinc that passes later is absorbed in the enterocytes, thereby upregulating the metallothionein, dissolved copper (e.g. free copper or weakly bound copper such as copper-containing tap water or other liquids, Without proper treatment by the enterocytes, the water flux inhibits the subsequent absorption of the liver (which enters the liver circulation and is bound to albumin). As a result, copper that is weakly bound to free copper or albumin at the main part of such portal veins enters the liver and then enters the serum circulation in a free or weakly bound state. As a result, oxidation occurs and adversely affects the patient's arteries, nerves and other organ systems, resulting in increased levels of atherosclerosis, dementia, mild cognitive impairment, Alzheimer's disease, Parkinson's disease, and free copper. Promotes other related diseases.

<Experiment 12>
Half-life radioisotopes Cu ⁶⁴ or Cu ⁶⁷ and / or stable copper isotopes Cu ⁶³ or Cu ⁶⁵ prepared as capsules or pills as described above and dissolved in water were orally administered to animals or humans. Using crossover experiments, natural copper (39% Cu ⁶³ and 69% Cu ⁶⁵ ) was orally administered dissolved in pills or water as described above. This administration period is short, up to 90 days, but longer for stable copper isotopes. A portal vein and / or serum sample is collected and fractionated into components using a column capable of separating ceruloplasmin, transcupriene, albumin, small protein and peptide. The copper isotope bound to each component is measured, and the isotope is administered in a protein-bound dosage form and the ceruloplasmin and transcuprine-bound copper isotopes are more statistical than those in the dissolved dosage form. The ratio was significantly large. Copper isotopes administered dissolved in water showed a high proportion of weakly bound albumin and other low kinetic binding proteins and amino acids. This result shows an improvement in pills or capsule preparations containing early-release copper and gastric zinc, which prevents the subsequent absorption of free copper dissolved in water. The tendency to hypocopperemia is reduced. This result is more pronounced for animals and patients with liver dysfunction (eg, patients with cirrhosis, hepatitis, reduced bile flow, primary sclerosing cholangitis or animal models and elderly patients). The clinical effect of this invention can be demonstrated by measuring the amount of plaque in an animal model of atherosclerosis and Alzheimer's disease (eg, Taconic rats) and conducting a memory test.

<Experiment 13>
This is an experiment to dissolve ATTM as an encapsulated preparation.
<Introduction>
Dissolution experiments were performed using capsules containing ammonium tetrathiomolybdate and 20 mg additive. The ATTM crystal used is less than 500 microns. ATTM was not recovered in the gastric juice dissolution medium at any time. After dissolution for 3 hours, suspension of black particles released from the capsule was observed in the solution. After neutralizing the gastric juice medium to pH 9, these black particles disappeared and about 10% of ATTM was recovered.

<Summary>
1. No ATTM dissolution was observed in the experimental solution.
2. After dissolution for 3 hours, about 10% undissolved ATTM remained in the experimental solution.
3. Conclusion: About 90% of ATTM was degraded in the stomach.

<Method>
The experimental materials are as follows.
1. The experiment was based on ATTM capsules prepared by R & D Pipex.
2. The ATTM was crushed and passed through a 500 micron sieve.
3. The capsule contains about 20 mg of ATTM and 180 mg of additive.

The dissolution conditions are as follows.
1. Medium: 1 liter of UPS artificial gastric fluid with pH 2 Temperature: 22 ° C
3. Paddle speed RPMs: 100
4). Dissolution time: 3 hours

The procedure is as follows.
1. QualiCaps ™ [Shionogi Europe, BV] # 2 capsules were mechanically loaded into the media at zero time.
2. After 1, 2, 4, 8, 16, 30, 45, 75, 120 and 180 minutes, 1 ml of medium was collected.
3. After 3 hours of dissolution, the pH was raised to 9 using NaOH.
4). The medium was paddle stirred for 1 hour.
5. After 1 hour, a sample was taken for analysis.

The analysis was performed as follows.
1. 400 μl of the collected medium was diluted to 2 ml with phosphate buffer pH 7.
2. The solution was placed in a cuvette and scanned from 260-600 nm.
3. Absorbance was measured at 367 nm.
4). The extinction count was calculated according to Beer's law.
5. The extinction count of the control sample in the capsule is 13500 cm ⁻¹ M ⁻¹ .

<Conclusion>
1. All ATTM dissolves in pH 2 media and degrades immediately.
2. After dissolution for 3 hours, a portion of ATTM floated in the medium as black granules.
3. After 3 hours, when the pH of the solution reached pH 9, the color of the solution changed to orange.
4). This is presumably because stable ATTM was dissolved.
5. The residual amount of ATTM was about 10% of the theoretical amount.
6). Therefore, 90% of the original dose of ATTM was degraded in the artificial gastric juice in 3 hours.
7). For enteric coatings, the dosage must be much smaller to achieve the same therapeutic effect.

表は、胃液の中でのATTMカプセルの溶解状況を示している。表中、“Ｕ”は未溶解ATTM、“Ｔ”は理論上100％未溶解ATTMである。

The table shows the dissolution status of ATTM capsules in gastric juice. In the table, “U” is undissolved ATTM and “T” is theoretically 100% undissolved ATTM.

<Experiment 14>
The stability of the ATTM capsule after 8 weeks was examined.
<Introduction>
The stability was examined using capsules prepared with ammonium tetrathiomolybdate. The test was conducted under various conditions. The best results were obtained when QualiCaps ™ (low moisture methylcellulose) was used, nitrogen was used in the storage atmosphere, and an additive containing lactose monohydrate was used.

<Summary>
1. ATTM was completely stable after 8 weeks storage at room temperature (RT) and ambient relative humidity (RH).
2. ATTM was 60-90% stable after 8 weeks storage at 40 ° C. and 70% RH.

The performance of the preparation at 40 ° C. and 70% RH is as follows.
1. QualiCaps capsules were better than gel cups (4 of 4 cases).
2. Nitrogen was better than air (3 of 4 cases).
3. Lactose monohydrate was better than anhydrous lactose (3 of 4 cases).

<Conclusion>
1. The best preparation conditions were QualiCaps capsules, nitrogen and lactose monohydrate.
2. The worst preparation conditions were gel cup, air and anhydrous lactose.

ATTM調製物の安定性を室温及び周囲相対湿度の条件で試験した。表中、“Mono”は一水和物ラクトース、“Anhydrous”は無水ラクトース、“Quali”はメチルセルロースカプセル、“Air”は大気雰囲気、“N2”は窒素雰囲気を表す。

The stability of the ATTM preparation was tested at room temperature and ambient relative humidity conditions. In the table, “Mono” represents monohydrate lactose, “Anhydrous” represents anhydrous lactose, “Quali” represents a methylcellulose capsule, “Air” represents an air atmosphere, and “N2” represents a nitrogen atmosphere.

  With respect to ammonium tetrathiomolybdate (ATTM) or ammonium polysaccharide (API), preparations and capsules containing them can be prepared in various ways and are described below.

<Production of ammonium tetrathiomolybdate (ATTM) powder>
1. A method of separating ATTM from oxidized forms of active ingredients that appear as black tar-like by-products (flaked tar) by milling ATTM under conditions of low moisture and low oxygen.
2. One method in which ATTM granulation produces a powder ATTM with an average particle size reduced to a size that can be separated from flake-like tar-like by-products.
3. ATTM is a method of sieving to produce ATTM powder and flaky tar.
4). A method wherein the sieving is selected such that the ATTM powder and flaky tar can be separated.
5. A method in which ATTM powder is separated from flaky tar and fine ATTM particles are separated from coarse flaky tar in a vessel in which vibration is used.
6). A method in which fine ATTM particles can be separated and collected from coarse flaky tars by mechanical vibration and gravity.
7). 6. The process of 6 wherein the coarse flaky tar layer is removed from the fine ATTM particle layer.
8). A method of milling ATTM under conditions of low moisture and low oxygen so that ATTM powder separated from tar is ground into ATTM particles with small average particles.
9. 2. Method 2 in which ATTM with a small average particle size dissolves rapidly in an aqueous solvent or gastrointestinal environment.
10. The ATTM granulation is performed using a mechanical device, mechanical (hand sieve, etc.) or power drive (blender, etc.), and the average particle size of the obtained ATTM particles is smaller than the original size. .
11. 10 ways to produce particles with an average particle size smaller than 100 microns by milling.
12 10 methods for producing particles having an average particle size of less than 10 microns by milling.
13. 10 methods to produce particles with an average particle size smaller than 1 micron by milling.

<Selection of additive>
14 A method of preparation by mixing ammonium polysaccharide (API) with additives that enhance the pharmaceutical properties of the API.
15. Additives 14 preparations that limit the moisture content of the API.
16. Examples are, but not limited to, 15 examples that are water lactose.
17. Additives 14 preparations that limit API oxidation.
18. Illustrative examples include, but are not limited to, 17 illustrative examples of crown ethers.
19. 14 additives to limit the reaction of reactive oxygen and / or nitrogen with API.
19 examples containing 20.1 or more free radical scavengers or antioxidants (including but not limited to mannose, butylated hydroxytoluene, etc.).

<Selection of salt>
21. API is a preparation method that generates cations and salts to improve or protect the pharmaceutical properties of API.
22. 21. The method according to 21, wherein the cation is an antioxidant.
23. Illustrative examples include, but are not limited to, 22 illustrative examples of nicotine esters of tocopherol.
24. Preparation of 21 in which the cation is hydrophobic and has the effect of preventing the hydration of the API.
25. Illustrative examples include, but are not limited to, 24 illustrative examples of benzylamine.

<Preparation added to additives to prevent acid hydrolysis of API>
26. A preparation method in which the formulation as part of the additive is added to the API to prevent acid hydrolysis of the API.
27. Preparation of 26, where the gastric acid hydrolysis of API is prevented by the addition of anti-acid additives.
28. 26 preparations wherein the antacid is administered separately from the ATTM pharmaceutical preparation, shortly before administration of the ATTM pharmaceutical preparation, at the same time as administration of the preparation, or shortly after administration of the preparation.
29. Examples include, but are not limited to, 27 examples of sodium bicarbonate.
30. Examples include, but are not limited to, 28 examples of sodium bicarbonate.
31. 26 preparations where gastric acid hydrolysis of the API is hindered by complexation into the formulation added to the additive.
32. 26. When API is dissolved in gastric juice, API is complexed with a formulation added to additives that bind API and prevent acid hydrolysis and oxidation of API.
33. Examples are, but not limited to, aminopolysaccharides (APIs), and examples of aminopolysaccharides are 32 examples which are polyglucosamine or chitosan.
34. Chitosan added to the additive, after dissolving in gastric juice, forms an insoluble colloidal complex with API, thereby protecting the API from acid hydrolysis 33 mechanisms.
35. A preparation in which part or all of the ATTM is enteric coated and the dissolution of the active ingredient is delayed until the acidic environment of the stomach is neutralized or until the part of the gastrointestinal tract where ATTM is neutralized is reached. object.
36. 35 methods wherein some or all of the API is enteric coated as a microtablet encapsulated in a capsule.
37. The enteric coating can be prepared in 35 ways that can change the dissolution rate of the microtablets.
38. 35 methods wherein one tablet to be administered is enteric coated.
39. 35 methods wherein tablets of various enteric coatings are prepared such that when administered to a patient, API is released from the enteric coating at different rates.
40. 35. The method of 35, wherein the enteric coating is administered with a copper-lowering agent that is not enteric coated.
41. 40 methods wherein the copper lowering agent is, but not limited to, zinc acetate, zinc sulfate, tetrathiomolybdate, tetrathiotungstate, penecillamine, or trientine.

<Formulation added to the additive to assist API absorption>
42. A preparation method in which the formulation as part of the additive is added to the API so that a complex is formed that enhances the absorption of the API.
43. Examples are, but not limited to, aminopolysaccharides, and examples of aminopolysaccharides are 42 examples that are polyglucosamine or chitosan.
44. The chitosan added to the additive is dissolved in the gastric juice, and then forms an insoluble colloidal complex with API, which is metabolically converted to API and a disaccharide or trisaccharide complex to become a suitable substrate for absorption. Mechanism.

<API / additive encapsulation material>
45. An API / additive encapsulation method wherein a capsule material is selected that enhances the pharmaceutical properties of the API.
46. The capsule material is 45 encapsulated to prevent API hydration.
47. The capsule material is 45 encapsulated with very little water content.
48. The encapsulant is 45 encapsulation that is leakproof under acidic conditions.
49. The encapsulant is 45 encapsulated, which is highly soluble only under acidic conditions.
50. The encapsulant is 45 encapsulated, which is highly soluble only under neutral conditions.

<Coating of capsules or tablets targeting API dissolution in specific organs>
51. A preparation method in which a coating is used to dissolve an API targeting a specific organ of the GI (gastrointestinal) tract.
52. 51. A coating method according to 51, wherein the API is prevented from dissolving in the oral cavity but immediately dissolved in the stomach.
53. 51. A coating method according to 51, in which API is prevented from dissolving in the stomach but dissolves immediately in the upper or lower part of the intestine.

<Dissolution of API targeting multiple specific organs>
54. A preparation method in which capsules and coatings are used to release API in all organs of the GI tract with a single administration of API.
55. 54 preparations utilizing multiple release mechanisms.
56. API is an example of 55 released in the stomach targeting the stomach. For dissolution in the stomach, it is preferable to complex copper from foods and drinks in the stomach so that copper is not absorbed in the intestine.
57. The fine API powder and additives contained in the capsule are 56 examples that dissolve immediately in gastric acid. The additive contains an API lowering agent that prevents hydrolysis but acts to allow the API to interact with free copper in the stomach.
58. 55 preparations where a portion of the API formulation is packaged to be dissolved in the intestine.
59. A portion of the API formulation 55 preparations coated so that degradation takes place in the intestine.
60. Preparation of 55 capsules containing API powder and additives that dissolve in the stomach. Capsules contain tablets that are coated for dissolution in the intestine. Thus, API dissolution in the stomach is used to bind copper, preventing copper absorption, API dissolution in the intestine, API is absorbed systemically, copper is absorbed systemically Can do.

<Co-preparation of API with other natural products known to complex copper>
61. A method of preparation in which natural products known to complex copper are added to the additive.
62. The example is, but not limited to, sodium alginate. Powdered sodium alginate is a common food known to bind heavy metals and prevent their systemic absorption.
63. By using sodium alginate, it is possible to bind free copper from food in the stomach and dissolve the coated API in the intestine to be absorbed systemically.

<Preparation of API that enables sustained sustained release>
64. A preparation method for sustained release of API in the stomach.
65. 64 preparations mixed with a formulation that is semi-resistant to dissolution in gastric acid and has an overall material density that results in buoyancy in gastric juice.
66. Examples are, but not limited to, 65 examples where API is prepared in digestible wax. Wax density provides buoyancy in gastric juice, and the slow digestibility of the wax results in sustained release of the API.

<Features of function>
67. A method for reducing the number of copper-lowering therapeutic agents that are orally administered to patients.
68. A method of increasing the absorption of API by administering ATTM to patients.
69. A method of administering an ATTM preparation to a patient to increase the pharmacological effect of the formulation at the same dose.
70. A method of achieving signs of serum copper, serum ceruloplasmin, urine copper or hepatic copper reduction by administration of a small dose of ATTM preparation.
71. How to achieve faster signs of copper loss by administering the same dose of ATTM preparation.

<Monitoring and administration>
72. A method in which a patient or caregiver monitors the effectiveness of an ATTM-containing preparation or a dosing program by evaluating body fluids, excretion or organs as therapeutic markers that indicate effective copper-lowering treatment.
73. 72 methods wherein the body fluid is urine.
74. 72 methods wherein the body fluid is blood or serum.
75. 72 methods wherein the body fluid is plasma.
76. 72 methods wherein the body fluid is saliva.
77. 72 methods wherein the bodily fluid is cerebrospinal fluid.
78. 72 methods in which the material evaluated is stool.
79. 72 methods in which the material evaluated is liver.
80. Longitudinal imaging methods (e.g., but not limited to proton magnetic resonance imaging, magnetic resonance imaging, brain or other organ computer topography, etc.) indicate slowing or regression of disease progression 72 methods.
81. 72. A method in which a patient's cognitive assessment over time is made to indicate a delay or regression in disease progression.
82. 72 Methods of assessing a patient's quality of life are made to indicate a delay or regression of disease progression.

<Disease treatment>
83. The preparation is one method used to treat a disease required for copper reduction therapy.
84. The preparation is 84 methods used to treat or prevent central nervous disease, inflammatory disease, disease in which fibrosis is a progression factor, or blood vessel-derived disease (including cancer).
85. 84 methods wherein the central nervous disease is, but is not limited to, Wilson's disease, Alzheimer's disease, Huntington's disease, schizophrenia, Parkinson's disease, ALS and prion disease.
86. 84 methods wherein the inflammatory disease is, but is not limited to, psoriasis, rheumatoid arthritis, lupus, inflammatory bowel disease, and the like.
87. Fibrosis includes but is not limited to idiopathic pulmonary fibrosis, primary biliary cirrhosis, nonalcoholic steatohepatitis, cirrhosis, glomerulonephritis, systemic fibrosis, rheumatoid arthritis, postoperative adhesions, adults There are respiratory distress syndrome (ARDS), coal miner pneumoconiosis (CWP), Hermannsky-Padlac syndrome (HPS), systemic sclerosis (SS), tumor stroma in lung cancer and obstructive bronchitis (OB) after lung transplantation84 the method of.
88. 1. A method of treating mammalian acetaminophen overdose or poisoning.
89. 1. A method for preventing or treating copper overdose or poisoning in mammals.

<API preparation to form a protein / API complex. The complex is formed in the stomach, forms a complex with copper released from food into the stomach, is impervious to acid hydrolysates, and from the intestine. Is not excreted and absorbed into the bloodstream>
90. A preparation method in which protein is added as part of an additive.
91. 54 methods in which the protein added as part of the additive forms a stable complex with the API in the stomach.
92. A stable API-protein complex is a method of 54 that can complex copper in the stomach.
93. 54. A method by which a stable API-protein complex is prevented from being absorbed into the bloodstream when it passes through the stomach.
94. 54 methods in which a stable API-protein complex passes through the GI tract and is excreted along with feces.
95. 54 preparation examples in which bovine serum albumin is added to the additive.
96. 54 ways in which API and bovine serum albumin form a stable complex capable of complexing with copper in the stomach.
97. 54 preparation examples of a synthetic D-polypeptide complexing with copper, the complex forming a complex with copper and passing through the original tubule without being digested in the GI tract.
98. 61 mechanisms by which D-polypeptide passes through the GI tract as a complex with API and copper without being digested.

<Method for preparing purified API>
99. A method of preparing purified ATTM by lyophilization after lysis.
100. 99 methods in which ATTM is dissolved in distilled spring water and purged with an inert gas (eg, nitrogen, argon, etc.).
101. 99 methods in which ATTM is dissolved in water, purged with an inert gas, and filtered through a 0.22 micron (or smaller) sterilizing filter.
102. The ATTM filter aqueous solution is poured into a cooled bottle (eg, -80 ° C.) and then swirled to form a frozen film on the inner surface of the bottle.
103. 99 methods in which the contents of the bottle are lyophilized.
104. 99 methods of filling empty bottles with pure dry argon or dry nitrogen after lyophilization.
105. 99 methods in which ATTM is collected and stored under inert gas.

<Method of tableting freeze-dried ATTM>
106. Lyophilized ATTM is tableted as the neat material of the preparation.
107. 106 methods in which tableted ATTM provides sustained release as a pharmaceutical property.
108. 106. A method in which tableted ATTM provides pharmaceutical properties (eg, enteric coatings) that are released into the target organ.

  Although described with respect to treatment of excess metal accumulation or metal malabsorption in animals or humans, other applications are possible. For example, the advantages of selection, sustained or delayed release by controlled crystal size have been described with respect to ATTM, but can also be used effectively for controlled delivery of other active formulations.

Claims (213)

  A pharmaceutical composition comprising a sustained release preparation of a formulation that induces inhibition of copper absorption in animals or humans.   The pharmaceutical composition of claim 1, wherein the composition is prepared such that release of the formulation begins in the small intestine or upper jejunum.   The composition of claim 2 in a coating that delays release.   The pharmaceutical composition of claim 1, wherein the formulation is zinc, preferably a zinc-cysteine complex, more preferably zinc-monocysteine.   2. The pharmaceutical composition of claim 1 wherein the formulation is a zinc salt.   The pharmaceutical composition of claim 5, wherein the zinc salt is selected from the group consisting of zinc acetate, zinc carbonate, zinc sulfate, zinc gluconate, zinc oxide, zinc chloride and zinc stearate.   The pharmaceutical composition of claim 4, wherein the dosage of zinc is 25 mg to 150 mg.   The pharmaceutical composition of claim 4, wherein the zinc is released over a period of 30 minutes to 24 hours.   A pharmaceutical composition comprising an injectable preparation for sustained release of zinc, preferably a zinc-cysteine complex.   The pharmaceutical composition of claim 9, wherein the zinc is released in 7 to 30 days.   A sustained release transdermal patch comprising zinc, preferably a zinc-cysteine complex, wherein the zinc is delivered through the skin.   A gel comprising zinc, preferably a zinc-cysteine complex, and an absorption enhancing additive capable of delivering zinc through the skin.   A subcutaneous implant capable of continuously releasing zinc, preferably a zinc-cysteine complex, for at least 30 days or more.   A subcutaneous implantable pump capable of continuously releasing zinc, preferably zinc-cysteine complex, for at least 30 days or more.   A gastric retention pill capable of slow release of zinc, preferably a zinc-cysteine complex, in the upper gastrointestinal system.   A gastrointestinal implantable device capable of slow release of zinc, preferably a zinc-cysteine complex, in the upper gastrointestinal system.   A preparation that is taken with food containing zinc, preferably a zinc-cysteine complex, such that the zinc competitively inhibits the absorption of copper from the food and is optionally a powder.   A preparation which does not break down in beverages and contains more than 50 mg of zinc, preferably a zinc-cysteine complex, in a single dose, optionally a powder.   A pill, tablet or capsule which does not break down in beverages and contains more than 50 mg of zinc, preferably zinc-cysteine complex, in a single dose.   A deionized beverage comprising 50 mg or more of zinc, preferably zinc-cysteine complex, in a single dose.   A pill, tablet or capsule comprising a cholesterol lowering drug and zinc, preferably a zinc-cysteine complex.   An orally administrable high dose sustained release preparation comprising zinc, preferably a zinc-cysteine complex.   23. The preparation of claim 22, wherein the dosage is greater than 100 mg.   An orally administrable pharmaceutical composition comprising a non-ionic form of copper.   25. The composition of claim 24, wherein the copper is complexed with a digestible protein.   It consists of two parts, the first part is the part released in the animal or human gastrointestinal tract and contains zinc, the second part after zinc induces the production of metallothionein in the gastrointestinal tract A pill, tablet or capsule that only contains a copper chelator, the part chelating copper.   A method of treating a disease characterized by excessive levels of homocysteine, comprising administering zinc, preferably a zinc-cysteine complex, and optionally a preparation of folic acid and zinc, preferably a zinc-cysteine complex, or folic acid And administering a copper chelator or copper complex.   A pharmaceutical composition comprising 1.0 mg zinc oxide in a single dose.   A method of treating a copper mediated disease in an animal or human comprising administering packaged tetrathiomolybdate under conditions of low humidity and hypoxia.   Contains zinc, preferably a zinc-cysteine complex, that dissolves or degrades at different rates at different locations in the animal or human gastrointestinal tract, optimizes the induction of matrix metallothionein in the gastrointestinal tract, and efficiently inhibits potential copper absorption Can be a tablet, pill or capsule.   A method of treating a copper mediated disease in an animal or human comprising administering an induced dose of a copper chelator and folic acid and / or zinc, or a zinc acetate complex.   32. The method of claim 31, wherein the chelating agent is ammonium tetrathiomolybdate.   A pharmaceutical composition comprising zinc, preferably a zinc-cysteine complex, and folic acid. A pellet comprising zinc, wherein the zinc is selected from the group consisting of zinc, zinc-cysteine complex, zinc acetate or other zinc salts, the density of the pellet is greater than 2 g / cm ³ , the pellet is of animal or human Pellets that can be held in stomach folds.   35. The pellet of claim 34, wherein the pellet includes an enteric coating and delays the release of its contents until the pellet finishes passage through the stomach.   36. The pellet of claim 35, wherein the pellet is coated with one or more layers of bioadhesive and / or mucoadhesive polymers.   A pharmaceutical composition comprising zinc, preferably a zinc-cysteine complex, and an iron supplement.   A method of treating schizophrenia, comprising administering a copper complex, a chelating agent, or an inhibitor, and folic acid and / or zinc or a zinc-cysteine complex.   A prodrug consisting of zinc, zinc-cysteine complex, tetrathiomolybdate, trientine, d-penicillamine or other copper chelating agent, copper complexing agent, or copper absorption inhibitor, Metals that are cleaved, released or activated in vivo, and selectively involved in elevated levels of ceruloplasmin-unbound copper or ceruloplasmin-unbound systemic copper in the patient's gastrointestinal tract or serum Prodrug compositions that depend on the presence and availability of proteins.   Prodrug consisting of zinc, zinc-cysteine complex, tetrathiomolybdate, trientine, d-penicillamine or other copper chelating agent, copper complexing agent, or copper absorption inhibitor, preferably preferential to the liver A prodrug that is preferentially released to the liver upon contact with the enzyme or protein expressed in the liver.   A selective copper chelator in a pharmaceutical preparation suitable for oral administration to a mammal, the chelator capable of chelating copper in the gastrointestinal tract of a mammal.   42. The chelator of claim 41, wherein the chelator has a systemic ecological utility of less than 2%.   A pill, tablet or capsule suitable for oral administration to animals or humans, comprising one or more microspheres, which can chelate copper in the mammalian gastrointestinal tract A pill, tablet or capsule containing a selective copper chelator.   A method for treating copper poisoning caused by decreased liver function by administering a copper chelator in an elderly animal or human.   45. The method of claim 44, further comprising administering an anti-inflammatory or anti-fibrotic formulation to promote copper excretion from the liver of an aged animal or human.   46. The method of claim 45, wherein the anti-inflammatory formulation is ursodiol.   A dietary supplement that is low in copper and high in zinc, preferably a zinc-cysteine complex.   A method of selecting a dose of a copper chelator by directly measuring copper not bound to ceruloplasmin in an animal or human.   A method of selecting a dose of a copper chelator by measuring the copper chaperone of Cu / Zn superoxide dismutase in animals or humans.   A preparation comprising enteric coated sustained release zinc and ascorbic acid.   51. The preparation of claim 50, further comprising folic acid.   A method for selecting a dose of a copper chelator, including determining the free copper available in the patient's body and selecting a maintenance dose that achieves a target range of free copper based on extrapolation. How to go.   A method of selecting a dose of a copper chelator that determines the free copper available in the patient's body by direct measurement of free copper in the serum and achieves a target range of free copper based on extrapolation Selecting a maintenance dose.   A method for selecting a dose of a copper chelator, wherein the serum level of a copper chaperone of Cu / Zn superoxide dismutase is determined by direct measurement of proteins in animal or human serum, saliva or urine, and extrapolation Selecting a maintenance dose to achieve a target range of free copper based on the method.   A method of selecting a dose of a copper chelator, measuring isoplastane levels in animal or human serum, saliva or urine and maintaining a target range of free copper based on extrapolation Selecting a dose.   A method of treating a condition of high homocysteine, comprising administering zinc.   A method of treating schizophrenia, Huntington's disease or amyotrophic lateral sclerosis, comprising administering zinc, preferably a zinc-cysteine complex.   A pharmaceutical composition comprising a copper supplement, wherein the copper supplement is complexed with a substance present in human or animal breast milk.   59. The composition of claim 58, wherein the substance is howay.   59. The composition of claim 58, wherein the substance is a lipid.   59. The composition of claim 58, wherein the substance is a protein.   59. The composition of claim 58, wherein the substance is casein.   59. The composition of claim 58, further comprising zinc, preferably a zinc-cysteine complex.   59. The composition of claim 58, further comprising a zinc salt.   59. The composition of claim 58, further comprising gastroretentive zinc particles. A composition comprising Cu ⁶³ complexed with a protein.   67. The composition of claim 66 further comprising zinc, preferably a zinc-cysteine complex. A composition comprising Cu ⁶⁵ complexed with a protein.   69. The composition of claim 68, further comprising zinc, preferably a zinc-cysteine complex.   A pill, tablet or capsule comprising zinc, preferably a zinc-cysteine complex, and a liver function agent, preferably a bile acid binder and / or a copper reducing agent.   The pill, tablet or capsule of claim 70, wherein the liver function agent is ursodiolic acid.   A tablet comprising zinc, preferably a zinc-cysteine complex, and a liver function agent, preferably a bile acid binder and / or a copper reducing agent.   The tablet of claim 72, wherein the liver function agent is ursodiolic acid.   A pharmaceutical composition comprising a stable copper isotope 65Cu or 67Cu in a ratio other than about 31% and about 69% and a liver function agent.   75. The pharmaceutical composition of claim 74, wherein the liver function agent is ursodiolic acid. A method for diagnosing a copper metabolic disorder, comprising administering Cu ⁶⁵ and Cu ⁶⁷ to a human or animal and measuring the proportion of Cu ⁶⁵ and Cu ⁶⁷ bound to at least one protein .   The method of ceruloplasmin 76, wherein the protein is selected from the group consisting of ceruloplasmin, trunk plain, superoxide dismutase, albumin and peptide.   77. The method of claim 76, wherein the protein is a high kinetic binding protein.   77. The method of claim 76, wherein the protein is a low kinetic binding protein. A method of modulating the treatment of a copper metabolic disorder, comprising administering Cu ⁶⁵ and Cu ⁶⁷ to a human or animal and measuring the proportion of Cu ⁶⁵ and Cu ⁶⁷ bound to at least one protein. How.   A method of treating a subject with Alzheimer's disease, comprising administering a composition that protects the subject from exposure to a soluble form of copper contained in a beverage.   82. The method of claim 81, wherein the composition comprises zinc, preferably a zinc-cysteine complex.   A method of treating a human or animal with Alzheimer's disease, comprising administering a complex of a high molecular weight natural protein and a biologic.   A pharmaceutically acceptable composition, suitable for humans or animals, comprising tetrathiomolybdate, wherein tetrathiomolybdate is less than 120 mg, optionally less than 100 mg, or less than 80 mg, Or less than 60 mg, or less than 40 mg, or less than 20 mg, or less than 10 mg, or less than 5 mg.   A method of treating a sick donor, comprising administering 2 μg to 20 mg of thiomolybdate daily.   A method of treating a human or animal with CNS disease comprising administering a formulation capable of stabilizing copper levels in serum.   Superoxide dismutase, ceruloplasmin, metallothionein, transferrin, amyloid beta, apoe4 and CCS, PEGylated form of superoxide dismutase or liposome, cumulative injection or other sustained release copper complexation Agent.   85. The composition of claim 84, wherein the tetrathiomolybdate is a dosage form of a sustained release preparation.   A supplement containing two or more trace metals selected from the group consisting of zinc, copper, iron, calcium, molybdenum and manganese in a sustained release preparation.   90. The composition of claim 86, wherein the trace metal is bound to a natural or synthetic digestible carrier that further limits release and digestion.   88. The composition of claim 87, wherein the carrier is selected from the group consisting of howay, plant fiber, dry milk, infant formula, metallothionein and transferrin.   The zinc is released in 1 to 24 hours, 2 to 24 hours, 3 to 24 hours, 4 to 24 hours, 5 to 24 hours, 6 to 24 hours, 8 to 24 hours or 12 to 24 hours. Pharmaceutical composition.   Tetrathiomolybdate preparations composed primarily of large crystals that allow protection of the stomach from the gastric juice environment.   Tetrathiomolyb is composed of a group of small crystals that allow the formation of complexes with copper in food ingredients and large crystals that protect against gastric juices in the stomach and dissolve in the small intestine. Dating preparation.   Tetrathiomolybdate preparations composed primarily of small crystals that allow the formation of complexes with copper in food ingredients.   Administration of tetrathiomolybdate mainly composed of large crystals, with or without food.   Administration of tetrathiomolybdate crystals with different size ranges, with or without food.   Administration of tetrathiomolybdate, mainly composed of small crystals, with or without food.   99. The method according to any one of claims 96 to 98, wherein the administration of tetrathiomolybdate is 0.01 to 4 mg / kg / day in a mammal.   A preparation for protecting thiomolybdate from exposure to the stomach environment.   A preparation comprising tetrathiomolybdate encapsulated by an enteric coating.   Tetrathiomolybdate is a preparation in which the solution is lyophilized and the resulting powder is compressed into tablets and applied with an enteric coating.   A preparation comprising a zinc-containing copper reducing agent and an enteric coated tetrathiomolybdate powder.   A preparation of tetrathiomolybdate and mineral oil that is released in the small intestine in an encapsulated enteric-coated preparation delivery system.   Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), mild cognitive impairment, dementia, Huntington's disease, Pick's disease, Behcet's disease, schizophrenia, bipolar disorder, depression, autism and multiple sclerosis A method of using an anti-copper agent or a copper absorption inhibitor to treat a human or animal disease selected from the group consisting of:   Anti-copper agent or copper absorption inhibitor is zinc, zinc-amino acid complex, zinc salt, non-zinc salt amino acid complex, zinc cysteine complex, non-zinc cysteine complex, zinc monocysteine, thiomolybdate, tetrathiomolybdate, tetrathio A method of ceruloplasmin 105 selected from the group consisting of ammonium molybdate, trientine, penicillamine, clioquinol, phytic acid, citric acid, deferasirox and combinations thereof.   The anti-copper agent or copper absorption inhibitor is used with a second formulation selected from the group consisting of an acetylcholinesterase inhibitor, an NMDA receptor antagonist, an antidepressant, an antipsychotic, a cholesterol-lowering agent, and combinations thereof. 105 methods.   Humans selected from the group consisting of diseases associated with abnormal accumulation of copper-related proteins in the body, juvenile and sporadic inclusion body myositis in the elderly, cardiovascular disease, atherosclerosis, cerebral infarction and peripheral vascular disease Alternatively, a method of using an anti-copper agent or a copper absorption inhibitor for treating animal diseases.   A pharmaceutical preparation suitable for oral administration in humans or animals, comprising a thiomolybdate having a crystal size of substantially 100 to 1,000 microns at the widest position of crystal size.   110. The composition of claim 109, wherein the average size of the thiomolybdate crystals is about 350 microns.   A pharmaceutical preparation suitable for oral administration in humans or animals, wherein the individual weight of the crystals is about 0.3 picogram.   A method of controlling the dissolution, release or absorption of an orally administered dosage form of thiomolybdate in the human or animal gastrointestinal tract by changing the size or distribution of the thiomolybdate crystals.   Sustained release preparation of thiomolybdate, including capsule tablet dosage forms, or other delivery dosage forms suitable for oral delivery with food or separately from food, crystal size from 50 microns Larger thiomolybdate preparations that are immediate release in the stomach.   Thiomolybdate is a crystal of a size large enough that more than 25% of the thiomolybdate material passes through the gastrointestinal tract as thiomolybdate and binds endogenous copper secreted into the jejunum and intestine via bile. 114. The composition of claim 113, wherein   Immediate release capsule containing fine particles coated so that thiomolybdate crystals that can bind copper secreted endogenously into the jejunum and intestine via copper and bile contained in food are dissolved in the intestine A pharmaceutical composition comprising   116. A method of treating a Wilson disease patient by administering an oral formulation according to any one of claims 109 to 115.   117. The method of claim 116, wherein the Wilson disease is a Wilson disease that exhibits neurological symptoms.   118. The method of claim 117, wherein the thiomolybdate is orally administered to the patient three times a day with a meal and three times a day separately from the meal.   Thiomolybdate is orally administered to patients 3 times a day with meals and once a day separately from meals before going to bed. The dose before bedtime is the total of 3 times a day administered with meals. 119. The method of claim 118, wherein the amount is equivalent to:   119. The method of claim 118, wherein the thiomolybdate administered orally with the meal is swallowed just prior to the first mouthful.   120. The method of claim 119, wherein thiomolybdate administered orally with a meal is swallowed just prior to the first mouthful.   119. The method of claim 118, wherein the thiomolybdate administered orally with the meal is swallowed at about the same time as the first mouthful.   120. The method of claim 119, wherein thiomolybdate administered orally with a meal is swallowed at about the same time as the first mouthful.   119. The method of claim 118, wherein the thiomolybdate administered orally with the meal is swallowed within 15 minutes from the first mouthful.   120. The method of claim 119, wherein the thiomolybdate administered orally with a meal is swallowed within 15 minutes from the first mouthful.   119. The method of claim 118, wherein the thiomolybdate administered orally with the meal is swallowed immediately after the meal.   120. The method of claim 119, wherein the thiomolybdate administered orally with the meal is swallowed immediately after the meal.   119. The method of claim 118, wherein the thiomolybdate administered orally with the meal is taken or sprinkled on the food or drink of the meal.   120. The method of claim 119, wherein the thiomolybdate administered orally with the meal is taken or sprinkled on the food or drink of the meal.   119. The method of claim 118, wherein the individual dose of thiomolybdate is about 18-22 mg per dose.   120. The method of claim 119, wherein the individual dose of thiomolybdate is about 18-22 mg per dose.   121. The method of claim 120, wherein the treatment is continued for about 8 weeks.   122. The method of claim 121, wherein the treatment is continued for about 8 weeks.   120. The method of claim 119, wherein treatment is continued for about 16 weeks, with a daily dose of 120 mg per day for the first 2 weeks and 60 mg per day for the following 14 weeks.   121. The method of claim 120, wherein the treatment is continued for about 16 weeks and the daily dosage is 120 mg per day for the first 2 weeks and 60 mg per day for the following 14 weeks.   A method of reducing the incidence of leukopenia or anemia in a patient with Wilson disease who exhibits neurological symptoms and who is treated orally with thiomolybdate, wherein the patient's serum ceruloplasmin level is monitored at least once per month And when the patient's serum ceruloplasmin level is lower than 10 mg / dl, the dose is reduced.   Bottles or other containers that are substantially purged of oxygen with an inert gas and contain at least one desiccant and a capsule, tablet or other oral dosage form containing thiomolybdate suitable for oral administration A composite comprising   138. A method of improving shelf life and stability by refrigerated storage of the composite of claim 137 at about 4 ° C.   138. The method of claim 138, wherein the composite is held at room temperature prior to opening by the patient to reduce condensation and improve stability.   138. The method of claim 138, wherein the composite is held at room temperature after opening to reduce condensation and improve stability.   39. The composite of claim 38, wherein the composite is a capsule, tablet or other oral dosage form having a unit count of 6, 10, 12, 18, 24, 30, 36, 42, 48, 50, 96 or 100.   Enteric coated capsules containing immediate release zinc, zinc salt, zinc salt amino acid complex, zinc cysteine complex, non-zinc cysteine complex or zinc monocysteine, prepared to release its components after passage through the stomach, Tablet or other oral formulation.   A multivitamin preparation in which a redox active mineral containing copper or ions is complexed with digestible proteins, fibers or natural or synthetic substances so that such metals are less likely to enter a bolus into the patient's serum.   A multivitamin preparation in which a redox active mineral containing copper or ions is a dosage form of a sustained-release preparation, and such a metal is released so that it is difficult for a bolus to flow into a patient's serum.   Capsules containing thiomolybdate suitable for oral administration to humans, wherein thiomolybdate is released immediately in the stomach and the capsule has a water content of about 6% or less.   Capsules are methylcellulose carrageenan, polyester, poly (non-halogenated hydrocarbon), poly (halogenated hydrocarbon), poly (halogenated polyether), diene polymer, poly (high alkylene oxide, polyamide, polysiloxane, polysilane, Poly (acrylonitrile), poly (low alkylene oxide), acrylate polymer, polyacrylic acid and alternating copolymers, poly (ethylene terephthalate), poly (butylene terephthalate), poly (trimethylene terephthalate), poly (ethylene naphthalate), Polyethylene, polypropylene, polystyrene, polyisobutylene, polymethylpentene, poly (tetrafluoroethylene), poly (chlorotrifluoroethylene), poly (vinyl chloride), poly (vinyl fluoride), poly (vinyl bromide), poly (vinyl ion) ), Poly (vinylidene chloride), poly (vinylidene fluoride), poly (vinylidene bromide), poly (vinylidene iodide), chlorinated polyether, polybutadiene, poly (dicyclopentadiene), poly (butylene oxide), poly (Propylene oxide), poly (2,6-dimethylphenylene oxide), nylon, poly (dimethylsiloxane), poly (methylphenylsiloxane), poly (diphenylsiloxane), poly (methylphenylsilane), poly (acrylonitrile), poly (Ethylene oxide), poly (methyl acrylate), poly (ethyl acrylate), polymethacrylic acid, polyethylacrylic acid, poly (methyl methacrylate), poly (ethyl methacrylate), and alternating copolymers, block copolymers, random copolymers Polymer, graft copolymer, terpolymer, block ternary heavy Body, and a random terpolymer, and / or polyethylene, polypropylene, polystyrene, and composites of claim 145 comprising a material selected from the group consisting of any combination thereof.   Capsules containing thiomolybdate suitable for oral administration to humans, wherein thiomolybdate is released immediately in the stomach and the capsule is substantially free of moisture.   147. The composite of claim 146, wherein the composite is enteric coated with an impermeable material.   148. The composite of claim 147, wherein the composite is enteric coated with an impermeable material.   147. The composite of claim 146, comprising an additive having a moisture content of 1% or less.   148. The composite of claim 147, comprising an additive having a moisture content of 1% or less.   149. The composite of claim 148, comprising an additive having a moisture content of 1% or less.   150. The composite of claim 150, wherein the additive is anhydrous lactose.   152. The composite of claim 151, wherein the additive is anhydrous lactose.   153. The composite of claim 152, wherein the additive is lactose monohydrate.   The enteric coating is pH dependent and is selected to prevent coating degradation and zinc release while the pellet is in the low pH condition of the stomach and / or in the pylorus (pH 1.2-3.5). 35 pellets.   Enteric coatings include celluloses such as hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, methylcellulose, ethylcellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose succinate and carboxymethylcellulose sodium. Polymers; acrylic acid polymers and copolymers, preferably acrylic acid polymers and copolymers formed from acrylic acid, methacrylic acid, methyl acrylate, ammonio methyl acrylate, ethyl acrylate, methyl methacrylate and / or ethyl methacrylate ("Eudragit"); polyvinylpyrrolidone, poly And shellac pellet of claim 35 which is selected from the group consisting of (purified rack); vinyl acetate, polyvinyl acetate phthalate, vinyl polymers and copolymers such as copolymers vinyl acetate crotonic acid and ethylene-vinyl acetate copolymer.   19. A preparation according to claim 17 or 18, wherein the preparation further comprises an acetylcholinesterase inhibitor.   The preparation of claim 17 or 18, wherein the preparation further comprises an NMDA antagonist.   160. The preparation of claim 159, wherein the NMDA antagonist is memantine or flupirtine.   The preparation according to claim 17 or 18, wherein the preparation further comprises a preparation capable of improving liver function and bile clearance function.   164. The preparation of claim 161, wherein the formulation is a statin.   164. The preparation of claim 161, wherein the formulation is a statin.   164. The preparation of claim 161, wherein the formulation is tetrathiomolybdate.   30. The method of claim 29, wherein the disease is a neurological disease.   32. The method of claim 31, wherein the disease is a neurological disease.   Neurological diseases include Wilson's disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Menkes disease, progressive supranuclear palsy, dementia, mild cognitive impairment, autism, posterior intracranial Parkinson's disease, Pick's disease and 30. The method of claim 29, selected from the group consisting of schizophrenia.   Neurological diseases include Wilson's disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Menkes disease, progressive supranuclear palsy, dementia, mild cognitive impairment, autism, posterior intracranial Parkinson's disease, Pick's disease and 32. The method of claim 30, selected from the group consisting of schizophrenia.   30. The method of claim 29, wherein the disease is a neuromuscular disease.   32. The method of claim 31, wherein the disease is a neuromuscular disease.   32. The method of claim 31, wherein the neuromuscular disease is selected from the group consisting of juvenile and sporadic inclusion body myositis and elderly myositis.   30. The method of claim 29, wherein the neuromuscular disease is selected from the group consisting of juvenile and sporadic inclusion body myositis and elderly myositis.   32. The method of claim 31, wherein the disease is an autoimmune disease.   30. The method of claim 29, wherein the disease is an autoimmune disease.   32. The method of claim 31, wherein the disease is multiple sclerosis.   30. The method of claim 29, wherein the disease is multiple sclerosis.   32. The method of claim 31, wherein the disease is a cardiovascular disease.   30. The method of claim 29, wherein the disease is a cardiovascular disease.   32. The method of claim 31, wherein the cardiovascular disease is selected from the group consisting of atherosclerosis, cerebral infarction and peripheral vascular disease.   30. The method of claim 29, wherein the cardiovascular disease is selected from the group consisting of atherosclerosis, cerebral infarction and peripheral vascular disease.   32. The method of claim 31, wherein the disease is an inflammatory disease.   30. The method of claim 29, wherein the disease is an inflammatory disease.   32. The method of claim 31, wherein the disease is fibrosis.   30. The method of claim 29, wherein the disease is fibrosis.   32. The method of claim 31, wherein the disease is liver disease.   30. The method of claim 29, wherein the disease is liver disease.   32. The method of claim 31, wherein the liver disease is selected from the group consisting of non-alcoholic steatohepatitis and non-viral hepatitis.   30. The method of claim 29, wherein the liver disease is selected from the group consisting of non-alcoholic steatohepatitis and non-viral hepatitis.   32. The method of claim 31, wherein the disease is diabetes.   30. The method of claim 29, wherein the disease is diabetes.   32. The method of claim 31, wherein the disease is elderly-related copper excretion disorder.   30. The method of claim 29, wherein the disease is elderly-related copper excretion disorder.   A method of treating diurnal fluctuations in the level of free copper in serum and CNS, comprising: (a) administering a packaged tetrathiomolybdate under conditions of low humidity and hypoxia; (b) copper chelate Administering an inducing dose of an agent and folic acid and / or zinc, or a zinc acetate complex, wherein the copper chelator is preferably ammonium tetrathiomolybdate.   A method of maintaining healthy copper status in elderly patients, normalizing copper excretion in the liver by administering a copper chelator, and optionally anti-inflammatory to improve copper excretion from the liver in elderly patients Administering an anti-fibrotic formulation, wherein the anti-inflammatory formulation is preferably ursodiol.   86. The method of claim 85, wherein the thiomolybdate is selected from the group consisting of tetrathiomolybdate, trithiomolybdate, dithiomolybdate and monothiomolybdate.   90. The method of claim 86, wherein the formulation is tetrathiomolybdate.   90. The method of claim 86, wherein the formulation is a sustained release tetrathiomolybdate.   90. The method of claim 86, wherein the formulation is a cumulative injectable preparation of a copper binding agent bound to a peptide.   90. The method of claim 86, wherein the formulation is a PEGylated peptide comprising a cysteine residue.   200. The method of claim 199, wherein the PEGylated peptide is a PEGylated superoxide dismutase.   90. The supplement of claim 89, wherein the supplement further comprises a copper absorption inhibitor.   202. The supplement of claim 201, wherein the copper absorption inhibitor is zinc.   90. The supplement of claim 89, wherein the supplement is in a sustained release particulate or matrix.   Capsules containing thiomolybdate.   205. The capsule of claim 204, wherein the thiomolybdate is selected from the group consisting of tetrathiomolybdate, trithiomolybdate, dithiomolybdate and monothiomolybdate.   205. The method of claim 204, wherein the thiomolybdate is a thiomolybdate complex.   205. The method of claim 204, wherein the capsule is very low in moisture.   205. The method of claim 204, wherein the capsule contains an additive with very low moisture.   205. The method of claim 204, wherein the capsule is packaged in a conditioned packaging.   209. The method of claim 209, wherein the packaging is an impermeable foil pouch.   The method of claim 209, wherein the packaging is a blister pack.   209. The method of claim 209, wherein the packaging is purged and sealed with an inert gas.   213. The method of claim 212, wherein the gas is nitrogen or argon.